Expression of WW domain-containing oxidoreductase WWOX in pterygium.

PURPOSE: Pterygium was traditionally regarded as a degenerative disease, but certain characteristics suggest that pterygium is probably premalignant tissue. The human WWOX gene, encoding the WW domain containing oxidoreductase (WWOX, FOR, or WOX1), is a candidate tumor suppressor gene. In this study, we investigated the WWOX gene and protein expression in pterygium. METHODS: Pterygium tissues were obtained from patients (n=16, primary=8, recurrent=8) who received surgical excisions. Each tissue sample was further divided into head and body regions. The WWOX gene and protein expression were examined with immunohistochemistry, western blot, and quantitative PCR. For comparison, normal superior temporal bulbar conjunctivas were used as controls. RESULTS: Compared to the controls, upregulation of WWOX and its Tyr33 phosphorylation was observed in the head region of all pterygium specimens. In the head and body of the pterygium specimens, WWOX expression was significantly higher than in the controls. In addition, WWOX expression was stronger in recurrent pterygia than in primary pterygia. CONCLUSIONS: Increased WWOX expression, especially in the head region, is probably due to the invasiveness of the pterygium. Our results indicate that WWOX may play a role in pterygium progression and recurrence.

Prolidase Enzyme Activity in Conjunctiva and Pterygium Tissues.

BACKGROUND: The aim of this study was to determine prolidase activity in conjunctival tissue and its relationship with pterygium. MATERIAL AND METHODS: Prolidase activity was measured in 23 pterygium and 25 healthy conjunctival tissues and the 2 groups were compared. RESULTS: Prolidase enzyme activity could not be measured in either the healthy conjunctival or in pterygium tissues. The mean serum prolidase levels of the control and pterygium groups were 967.46±353.64 and 858.29±301.83, respectively. Statistically, there was no significant difference between the groups with regard to serum prolidase levels (p>0.05). CONCLUSIONS: In conclusion, absence of prolidase activity in pterygium tissue indicates that there is no collagen turnover in this tissue. We may explain this finding with the elastin-rich structure of the conjunctiva.

Overexpression of peroxiredoxin 2 in pterygium. A proteomic approach.

Pterygium is one of the most frequent pathologies in ophthalmology, and is a benign, fibrovascular lesion originating from the bulbar conjunctiva. It is composed of an epithelium and highly vascular, subepithelial, loose connective tissue. The etiology of pterygium is not clearly understood; the most widely recognized originating factor is ultraviolet radiation. It has been proposed that pterygium and neoplasia have common features, raising the possibility that pterygium is a neoplastic-like growth disorder. In this study, proteomic analysis was performed to show that peroxiredoxin 2 is overexpressed in pterygia compared to healthy conjunctivas. Twelve pterygium specimens were obtained together with healthy conjunctival tissue from the same eyes. Total proteins of pterygia and healthy conjunctivas were analyzed in SDS-PAGE. This analysis showed protein bands expressed exclusively in pterygium samples at the range of 20-25 kDa. After this, 2D electrophoresis was performed for the separation of total proteins; differential spots expressed in pterygium were excised and sequenced. Mass spectrometry (MS) data were searched in the NCBInr and EST databases using the MASCOT program. The spot was identified as peroxiredoxin 2. Real-time PCR, western blot and immunohistochemistry showed that peroxiredoxin 2 was increased in pterygium compared to healthy conjunctiva. Although, these results suggest that overexpression of peroxiredoxin 2 in pterygium could protect the cell against oxidative stress-induced apoptosis, further studies are required to establish the functional role of peroxiredoxin 2 in pterygium to determine its role in peroxidation and apoptosis in this pathology.

An association between BPDE-like DNA adduct levels and CYP1A1 and GSTM1 polymorphisma in pterygium.

PURPOSE: Benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), an ultimate metabolite of benzo[a]pyrene, attacks deoxyguanosine to form a BPDE-N2-dG adduct resulting in p53 mutations. Both cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1) have been demonstrated to be involved in the metabolism of polycyclic aromatic hydrocarbons. The relationship between BPDE-like DNA adduct levels and CYP1A1 and GSTM1 gene polymorphisms in pterygium is not clear. Therefore, BPDE-like DNA adducts and CYP1A1 and GSTM1 polymorphisms were detected in this study to provide more molecular evidence to understand the cause of BPDE-like DNA adduct formation in pterygium. METHODS: In this study, immunohistochemical staining using a polyclonal antibody on BPDE-like DNA adducts was performed on 103 pterygial specimens. For the analysis of CYP1A1 and GSTM1 polymorphisms, DNA samples were extracted from epithelial cells and then subjected to restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) for the determination of mutation and genotype of CYP1A1 and GSTM1. RESULTS: BPDE-like DNA adducts were detected in 33.0% (34/103) of the pterygium samples. The differences in DNA adduct levels were associated with the genetic polymorphisms of CYP1A1 but not GSTM1. Additionally, the risk of BPDE-like DNA adduct formation for patients with CYP1A1 m1/m2 (C/T) andm2/m2 (T/T) was 9.675 fold higher than that of patients with CYP1A1 m1/m1 (C/C) types (p=0.001, 95% Confidence Interval 2.451-38.185). CONCLUSIONS: Our data provide evidence that the BPDE-like DNA adduct formation in pterygium samples was associated with CYP1A1 polymorphisms.

The Significant Association of MMP-1 Genotypes With Taiwan Pterygium.

BACKGROUND/AIM: Few studies have examined the contribution of matrix metalloproteinases (MMP) to either diagnosis or prognosis of pterygium. The aim of this study was to investigate the contribution of MMP-1 genotypes to pterygium risk. PATIENTS AND METHODS: A total of 134 cases and 268 controls were included and their MMP-1 -1607 (rs1799705) genotypes were examined by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The percentages of 2G/2G, 1G/2G, and 1G/1G for rs1799705 genotypes were 48.5, 36.6 and 14.9% among patients and 33.9, 44.8, and 21.3% among controls (p trend=0.0167). The odds ratios (ORs) after adjusting for age and gender for 1G/2G and 1G/1G genotypes at rs1799705 were 0.54 (95%CI=0.33-0.89, p=0.0168) and 0.46 (95%CI=0.27-0.88, p=0.0192), respectively. Consistently, the adjusted OR for those carrying the 1G allele at MMP-1 -1607 was 0.61 (95%CI=0.41-0.78, p=0.0167), compared with the wild-type 2G allele. CONCLUSION: The genotypes at rs1799705 play a role in determining personal susceptibility to pterygium.

Real-time fluorescent quantitative polymerase chain reaction study of polo-like kinase-1 in pterygia.

This study was designed to examine the expression of polo-like kinase 1 (PLK1) mRNA in 16 pterygia and 13 normal conjunctival tissue specimens using real-time fluorescent quantitative polymerase chain reaction (PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. The difference in threshold cycle value (DeltaC(t)) was derived for PLK1 and GAPDH for each sample assayed, and the difference between the paired samples (DeltaDeltaC(t)) was calculated. The mean +/- SD DeltaC(t) of PLK1 mRNA was 9.56 +/- 1.30 in pterygia compared with 10.71 +/- 1.39 in normal conjunctiva. The expression of PLK1 mRNA in pterygium was 2.08 - 2.36 times that in normal conjunctiva; this difference was statistically significant. Real-time fluorescent quantitative PCR analysis appears to be effective and sensitive when determining the level of PLK1 mRNA expression. Using this method, it was demonstrated that PLK1 mRNA is over-expressed in pterygia, indicating a probable role for PLK1 in their development.

Cyclooxygenase-2 expression in primary and recurrent pterygium.

BACKGROUND: Pterygia are common, benign, fibrovascular, and infiltrative processes of the corneo-conjunctival junction of unknown pathogenesis. Cyclooxygenase-2 (COX-2) mediates the rate-limiting step in arachidonic acid metabolism. Extensive evidence indicates that the COX-2 prostanoid pathway is involved in inflammation. The aim of the study was to document the immunohistochemical expression of COX-2 in primary and recurrent pterygia. MATERIALS AND METHODS: In this study, 21 primary pterygia and 12 recurrent pterygia from subjects undergoing pterygium surgery and six normal corneal-scleral tissue specimens were studied immunohistochemically for COX-2 expression. RESULTS: COX-2 was expressed in primary pterygia and recurrent pterygia specimens. There was a statistically significant difference in COX-2 expressions in fibroblasts between primary and recurrent pterygium cases ( P = 0.001). There were statistically significant differences in COX-2 expressions in surface epithelium ( P = 0.028) and stromal inflammatory cells ( P =0.000) between control tissues and primary pterygia tissues. We also detected statistically significant differences in COX-2 expressions in surface epithelium ( P =0.000), stromal fibroblasts P =0.000 (stromal fibroblasts and inflammatory cells), vessels ( P = 0.027) and inflammatory cells ( P =0.001) between control tissues and recurrent pterygia tissues. CONCLUSIONS: This is the first study to document the expression of COX-2 in primary and recurrent pterygia. In our opinion after excision of pterygia, fibroblastic proliferation continues and this contributes to recurrence.

[Etiopathogenic aspects in development and evolution of pterigyum]. / Aspecte etiopatogenice ale dezvoltarii si evolutiei pterigionului.

Pterygium is an epithelial hyperplasia accompanied by a fibrovascular growth originating at the corneo-conjunctival junction, from where the modified limbic cells migrate and surpass the cornea. The studies reviewed show that it is an active process associated with cell growth, remodelling of the connective tissue, angiogenesis and inflammation. Despite the lack of knowledge regarding the pathogenesis of pterygia, epidemiologic evidence suggests that exposure to UV-irradiation may be an initial trigger in the development of this lesion. Other theories include changes of the apoptotic pathway the presence of some active angiogenetic factors or involvement of the MMPs, cytokines and growths factors. UV light could be the initial trigger that activates epithelial cells at or near the limbus to produce cytokines such as IL-6 and IL-8 and growth factors. These multifactorial proteins set up a cascade of events that include inflammation, proliferation, angiogenesis and antiapoptosis. Cytokines are able to induce the expression of MMPs and their tissue inhibitors (TIMPs) making it likely that they would also affect the rate of tissue remodeling, such as destruction of Bowman's membrane and the invasion of pterygium. In the etiology of pterygium abnormalities in tear functions have also been emphasized.

The Association of MMP-8 Genotypes with Pterygium.

BACKGROUND/AIM: Pterygium is composed of proliferating fibrovascular tissue, and its formation and progression are closely related to the homeostasis of the extracellular microenvironment. However, few studies have examined the contribution of matrix metalloproteinases (MMP) to either diagnostic or prognostic potential in pterygium. In this study, we investigated the contribution of a polymorphism in the promoter region of MMP-8 (-799C/T) and two non-synonymous polymorphisms (Val436Ala and Lys460Thr) to pterygium. MATERIALS AND METHODS: In this study, 134 patients with pterygium and 268 non-cancer controls patients were collected and the MMP-8 -799C/T, Val436Ala and Lys460Thr polymorphic genotypes of each subject were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The results showed that the three polymorphisms investigated were not significantly associated with risk of pterygium. In addition, the stratified analysis showed that there was no interaction between MMP-8 genotype with age or gender on pterygium risk determination. CONCLUSION: Polymorphisms at MMP-8 -799C/T, Val436Ala and Lys460Thr may not mainly contribute to determining personal susceptibility to pterygium in the Taiwanese examined.

Cyclooxygenase 2 expression in pterygium.

PURPOSE: Following the recent discovery of an abnormal expression of the p53 gene in the epithelium in pterygium, some researchers felt that pterygium is a tumor rather than a degenerative disease. Ultraviolet (UV) radiation has been reported to be associated with pterygium formation, however the mechanism whereby UV induces uncontrolled proliferation in pterygial cells is unclear. Because cyclooxygenase 2 (COX 2) was reported to exist and play an important role in UV-related cutaneous carcinogenesis, it is logical to suspect that COX 2 existed in pterygium. This study was designed to investigate the expression of COX 2 in pterygium. METHODS: Immunohistochemical staining using a monoclonal antibody to COX 2 was performed on 90 pterygial specimens, 40 normal conjunctiva, and 5 normal limbus. RESULTS: In the pterygium group, 75 (83.3%) specimens stained positive for COX 2. The staining was limited to the cytoplasm of the epithelial layer and predominantly over the basal epithelial layer. No substantial staining was visible in the subepithelial fibrovascular layers. All specimens were negative in the normal conjunctiva and limbus group. CONCLUSIONS: The present study showed COX 2 existed in pterygium. Given the role of COX 2 in cutaneous tumor carcinogenesis, we suggest COX 2 may also play a role in pterygium formation. This study could be used as the basis for future surveys of the causal relationship between COX 2 and pterygium as well as the effect of COX 2 inhibitor in preventing primary or recurrent pterygium.

Cyclooxygenase-2 expression in primary and recurrent pterygium.

PURPOSE: Pterygium is a proliferative, inflammatory, and invasive ocular surface disease associated with excessive ultraviolet radiation exposure and has several tumor-like characteristics. Cyclooxygenase-2 (COX-2) is an inducible enzyme and recently increased expression of the enzyme was found in many cancers and premalign lesions. This study was conducted to identify the COX-2 expression in pterygium tissues. METHODS: Immunohistochemical staining using a primary antibody for COX-2 was performed on 30 specimens with primary pterygium (20 pterygium without recurrence and 10 pterygium which recurred during a 12-month follow-up), 11 specimens with recurrent pterygium, and 8 specimens of conjunctival tumor. As a control we used 10 specimens of normal conjunctiva. Extent and intensity of cytoplasmic and membranous staining in epithelial cells were evaluated. RESULTS: Higher expression of COX-2 was detected in conjunctival tumor (87.5%) specimens and recurrent pterygium specimens (72.7%) compared to the both normal conjunctiva (30%) and primary pterygium without recurrence (30%). COX-2 expression in primary pterygium tissues with recurrence (60%) was not different from primary pterygium without recurrence (p=0.114) and recurrent pterygium (p=0.537). However, recurrent pterygium tissues were found to express higher COX-2 than primary pterygium without recurrence (p=0.022). CONCLUSIONS: COX-2 expression is increased in recurrent pterygium tissues and COX-2 expression may be a marker for the prediction of recurrence.

A PPAR-Gamma Agonist Rosiglitazone Suppresses Fibrotic Response in Human Pterygium Fibroblasts by Modulating the p38 MAPK Pathway.

Purpose: Fibroblast activation may play an important role in pterygium progression. Synthetic peroxisome proliferator-activated receptor γ (PPAR-γ) ligands have been shown to be effective antifibrotic agents against transforming growth factor ß1 (TGF-ß1) induced fibrosis in several tissues. We aimed to investigate the antifibrotic effects of the PPAR-γ ligand rosiglitazone in pterygium fibroblasts and the underlying mechanisms. Methods: Profibrotic activation was induced by TGF-ß1 in primary cultured human pterygium fibroblasts and the effect of rosiglitazone treatment on α-smooth muscle actin (α-SMA), and extra cellular matrix proteins synthesis was detected by western blotting, real-time PCR, immunostaining, and flow cytometry. Pharmaceutical inhibition of PPAR-γ receptor was used to determine the dependency or otherwise of rosiglitazone's action on PPAR-γ signaling. Major signaling pathways downstream of TGF-ß1 were investigated by western blotting to assess their possible association with rosiglitazone's effect. Cell viability and apoptosis were investigated to assess drug-induced cytotoxicity, and the effect of rosiglitazone treatment on cell migration was further determined. Results: α-SMA and fibronectin synthesis induced by TGF-ß1 were suppressed by rosiglitazone treatment in a dose-dependent manner. Rosiglitazone also inhibited intrinsic TGF-ß1 expression. Smad2/3, ERK1/2, and P38 pathways were activated in response to TGF-ß1. Rosiglitazone suppressed TGF-ß1-induced P38 MAPK activation, while ERK1/2 and Smad2/3 signaling remained unaffected. The observed antifibrotic effect of rosiglitazone was not affected by the PPAR-γ antagonist GW9662, indicating it is not PPAR-γ dependent. Rosiglitazone also inhibited the proliferation and migration of pterygium fibroblasts. Conclusions: Rosiglitazone suppresses TGF-ß1-induced myofibroblast activation and extra cellular matrix synthesis in pterygium fibroblasts at least partly through the modulation of the p38 MAPK pathway.

Hypermethylation of the p16 gene promoter in pterygia and its association with the expression of DNA methyltransferase 3b.

PURPOSE: A pterygium has long been considered as a degenerative condition. After p53 protein was found to be abnormally expressed in the epithelium, researchers suggested that a pterygium may be a tumor, but additional evidence is required to support this hypothesis. Aberrant methylation of the p16 gene (CDKN2A) promoter and resultant gene silencing play important roles in the pathogenesis of many types of human cancers. The purpose of this study was to investigate hypermethylation of the p16 promoter in pterygia and the relationship between this hypermethylation and the expression of p16 and DNA methyltransferase 3b (DNMT3b) proteins. METHODS: We studied the methylation status of p16 and the expression of p16 and DNMT3b proteins by performing methylation-specific polymerase chain reaction and immunohistochemistry, respectively, in specimens of 129 pterygia and 16 normal conjunctiva. The results were statistically analyzed. RESULTS: Hypermethylation of the p16 gene promoter was detected in 21 (16.3%) of 129 pterygial specimens. Among them, 46 (35.7%) were positive for p16 protein expression, and 83 (64.3%) were negative. Staining for p16 was limited to the nuclei of the epithelial layer. We observed a significant reverse correlation between hypermethylation of the p16 promoter and the expression of p16 protein (p=0.006). Thirty-eight (29.5%) pterygial specimens were positive for DNMT3b protein expression, and 91 (70.5%) were negative. DNMT3b staining was limited to the nuclei of the epithelial layer. A significant correlation was found between hypermethylation of the p16 promoter and the expression of DNMT3b protein (p<0.001). CONCLUSIONS: The p16 gene promoter was hypermethylated in pterygia, and this hypermethylation was strongly linked to expression of the positive expression of DNMT3b protein and to the suppression of p16 protein. These data provided molecular evidence that methylation occurs in pterygia and that it may play a role in the their development.

Matrix metalloproteinase-9 expression in pterygium.

PURPOSE: To assess matrix metalloproteinase (MMP)-9 expression in pterygium lesion. METHODS: A prospective randomized clinical trial was done to evaluate the expression of matrix metalloproteinase in normal and in primary or recurrent pterygia in Tenon's capsule by immunohistochemical analysis and a computerized image analysis system. The data were submitted to statistical analysis. RESULTS: Matrix metalloproteinase expression showed no difference in normal Tenon's capsule and in primary or recurrent pterygia. CONCLUSION: The similar expression of the matrix metalloproteinase in normal Tenon's capsule and in primary or recurrent pterygia allowed us to conclude that matrix metalloproteinase is not implicated in the genesis or the recurrence of pterygium lesion.

Investigation of Glutathione S-Transferase Isoenzyme Protein Expression in Patients With Pterygium.

PURPOSE: We investigated glutathione S-transferase (GST) enzymes in terms of their potential effects on the pathogenesis of pterygium. METHODS: Twenty-six pterygium specimens and 15 normal conjunctival specimens of 15 control subjects were investigated. Expressions of GST (alpha, mu, pi, and theta) enzymes were assessed by immunohistochemical staining. A brown color in the cytoplasm and/or nuclei of epithelial cells was evaluated as positive staining for GST enzymes. For each antibody, the intensity of the reaction [negative (-), weak (1+), moderate (2+), or strong (3+)] was determined to describe the immunoreactions. RESULTS: The median age was 52 years in the both groups. There was no significant difference between the groups in terms of age, sex, and intraocular pressure measurements (P > 0.05 for all). Of the 26 pterygium specimens, 15 (57.7%) (8 weak, 4 moderate, and 3 strong staining) were identified with GST pi-1 (GSTP1) expression and 20 (76.9%) (12 weak, 7 moderate, and 1 strong staining) with GST theta-1 (GSTT1) expression. Of the 15 control specimens, 4 (26.7%) (4 weak staining) were identified with the GSTP1 expression, and 1 (6.7%) with GSTT1 expression. GSTP1 and GSTT1 expressions were significantly higher in the pterygium specimens than in the controls (P = 0.043, P < 0.001; respectively). None of tissue specimens had positive staining for GST mu-1 or GST alpha-1 in both groups (both; P = 1.00). CONCLUSIONS: The significant increase of GSTP1 and GSTT1 expressions in pterygium may be because of the increased activation of GST in response to excessive free radical formation from ultraviolet exposure to maintain antioxidant capacity in pterygium.

Active matrilysin (MMP-7) in human pterygia: potential role in angiogenesis.

PURPOSE: Pterygia are invasive, proliferative fibrovascular growths, with the matrix metalloproteinase (MMP) family of enzymes strongly implicated in the pathogenesis of these lesions. The purpose of this study was to determine the cellular distribution and activation status of matrilysin (MMP-7) in pterygia. METHODS: Resected pterygia (n = 8) and normal conjunctiva (n = 8) were sectioned and analyzed immunohistochemically with two different epitope-specific anti-MMP-7 monoclonal antibodies (Abs) which differentiate pro- and active MMP-7. The specificity of each Ab was confirmed by Western blot analysis of p-aminophenylmercuric acetate (APMA)-activated and latent recombinant MMP-7. Pterygia (n = 4) and autologous normal conjunctiva (n = 4) were placed in organ culture to determine the activation status of secreted MMP-7. RESULTS: Precursor and active forms of MMP-7 were detected in epithelial cells from both pterygia and normal conjunctiva. Intense immunoreactivity for pro- and active MMP-7 was also observed in the pterygium vasculature, but was essentially absent from conjunctival vessels. Pro-MMP-7 was also identified in the epithelial basement membrane and associated with matrix components in pterygia. The 141-7B2 Ab reacted with the 30-kDa latent MMP-7, and the IM47L Ab precipitated a 19-kDa active enzyme, thus confirming the differential specificity of each Ab. Pro- and active MMP-7 were increased 1.4- and 2.7-fold, respectively, in the supernatants from organ-cultured pterygia compared with conjunctiva. CONCLUSIONS: This study is the first to specifically localize an active MMP species in pterygia and strengthens the hypothesis that these enzymes are involved in the pathogenesis of this disease. The data also suggest that MMP-7 may play a significant role in the angiogenesis that characterizes this lesion. Future studies will be directed at determining whether targeting MMP activity may be useful for treatment of pterygia.

Telomerase activity and p53 expression in pterygia.

PURPOSE: To investigate tolomerase activity and p53 expression in pterygial tissue. METHODS: Pterygia tissue was obtained during excisional surgery fr om 35 eyes of 35 patients, and superior bulbar conjunctival tissue from the same eye was also sampled as control when possible. Fluorescence telomeric repeat amplification protocol was used to measure telomerase activity in whole pterygium samples from 9 cases and in the epithelium and stroma of pterygium from another 10 cases. p53 protein content was measured by enzyme-linked immunosorbent assay (ELISA) in tissues obtained from 7 eyes, as well as in epithelial cell suspensions collected by brush cytology in 8 eyes. Six samples were also analyzed for UV-specific mutations in the p53 gene by the single-strand conformation polymorphism technique and DNA sequencing. A conjunctival epithelial cell line was irradiated with sublethal levels of UV-B to investigate whether telomerase activity can be induced in vitro. RESULTS: In all, 63% of pterygia samples demonstrated telomerase activity, whereas all 10 paired conjunctival control samples were negative (P = 0.05, chi-square test). Of the 10 samples in which telomerase activity was measured separately in the epithelium and stroma of pterygia, 5 samples were positive in the epithelium, only 1 of which had activity in the stroma. Average telomerase activity in positive samples was 18.44 +/- 8.77 U/microg protein, compared with telomerase activity measured in a carcinoma in situ patient (33.73 U/microg), and in an immortalized conjunctival epithelial cell line (50.72 +/- 15.55 U/microg). Telomerase activity was not upregulated in this cell line by UV-B exposure. All 6 pterygia samples tested for p53 mutations did not reveal the UV-specific mutations in exons 5, 6, 7, or 8. No statistical significance was observed in the pterygium or conjunctiva p53 protein levels in epithelial cells collected by brush cytology, while p53 protein level was lower in pterygia when measured in whole tissue samples. CONCLUSIONS: Telomerase activity was detected in some pterygia, mostly in the epithelium. Pterygia was not associated with an increase in epithelial p53 protein content measured by ELISA.

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.

Regulation of collagenase, stromelysin, and urokinase-type plasminogen activator in primary pterygium body fibroblasts by inflammatory cytokines.

PURPOSE: To examine the expression patterns of extracellular matrix degrading enzymes in cultured primary pterygium body fibroblasts activated by cytokines and growth factors potentially derived from ocular surface epithelial cells and tears. METHODS: EGF, TGF-alpha, PDGF-BB, IL-1beta, bFGF, TGF-beta1, TNF-alpha, or IL-6 were added at 10 ng/ml to early passaged primary pterygium body fibroblasts (PBF) or normal human conjunctival fibroblasts (HJF) in a serum-free medium. Expression of transcripts and proteins of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, and uPA was determined by Northern hybridization, ELISA, and Western blotting, respectively. Gelatin and casein zymographies were performed in their serum-free conditioned media with or without enzyme inhibitors to determine the activity of MMP-2 and -3, respectively. RESULTS: IL-1beta and TNF-alpha dramatically increased the mRNA and protein expression of MMP-1 and MMP-3 in cultured PBF when compared to normal HJF and to their nonstimulated counterparts cultured in a serum-free medium. EGF and TGF-alpha also upregulated MMP-3 in PBF when compared to HJF. The transcript levels of MMP-2 were high but stable for the two cell types regardless of the cytokine treatment. Both TIMP-1 and TIMP-2 expressions were not influenced by the cell type or the cytokine treatment. MMP-9 was not expressed in either of these two types of fibroblasts. Both IL-1beta and TNF-alpha induced a significant decrease in uPA expression in PBF, whereas bFGF induced a slight increase in both HJF and PBF. CONCLUSIONS: Chronic inflammatory stimulation by IL-1beta and TNF-alpha, which potentially can be derived from the ocular surface and tears, may be responsible for increased expression of MMPs in cultured PBF. These data have clinical implications on progression of pterygium and recurrence associated with incomplete excision of primary PBF under the influence of ocular surface inflammation. Suppression of intraoperative and postoperative inflammation may be a new strategy to prevent pterygium recurrence.

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.