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.

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.

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.

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.

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.

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.

Peroxiredoxin I and II in human eyes: cellular distribution and association with pterygium and DNA damage.

Peroxiredoxin I and II are both 2-Cys members of the peroxiredoxin family of antioxidant enzymes and inactivate hydrogen peroxide. On western blotting, both enzymes appeared as 22-kD proteins and were present in the sclera, retina and iris. Immunohistochemistry showed strong cytoplasmic labeling in the basal cells of the corneal epithelial layer and the corneoscleral limbus. The melanocytes within the stroma of the iris and the anterior epithelial cells of the lens also showed strong cytoplasmic labeling. The fibrous structure of the stroma and the posterior surface of the ciliary body were also labeled. There was also strong labeling for both enzymes in the photoreceptors and the inner and outer plexiform layers of the retina. There was increased labeling of peroxiredoxin I and II in pterygium. In normal conjunctiva and cornea, only the basal cell layer showed labeling for peroxiredoxin I and II, whereas, in pterygia, there was strong cytoplasmic labeling in most cells involving the full thickness of the epithelium. Co-localization of the DNA oxidation product 8-hydroxy-2'-deoxyguanosine antibody with the nuclear dye 4',6'-diamidino-2-phenylindole dihydrochloride indicated that the majority of the oxidative damage was cytoplasmic; this suggested that the mitochondrial DNA was most affected by the UV radiation in this condition.

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.

Ultraviolet-A irradiation upregulated urokinase-type plasminogen activator in pterygium fibroblasts through ERK and JNK pathways.

PURPOSE: Effects of ultraviolet (UV) B on the pterygium and its cells have been studied previously, whereas little is known on the effects of UVA. Urokinase-type plasminogen activator (uPA) is a protease involved in tissue remodeling and cell migration, and its levels are increased in pterygium. The purpose of our study was to investigate the effects of UVA on the expression of uPA in cultured pterygium fibroblasts. METHODS: Cultured fibroblasts from early-stage pterygia and normal conjunctiva were irradiated with different dosages of UVA and compared to nonirradiated cells. uPA activities in the medium and uPA mRNA in the cells were measured by casein zymography and RT-PCR, respectively. Total and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-related kinase (ERK), and c-Jun N-terminal kinase (JNK) levels of cells treated with and without UVA were measured by Western blotting. Inhibitors of p38 (SB203580), ERK (UO1026), and JNK (SP600125) were added before the irradiation of UVA to test their effects. RESULTS: UVA irradiation increased the uPA mRNA levels in pterygium fibroblasts and the uPA activities in cultured medium, which was accompanied with an increase in phosphorylated ERK and JNK. The ERK and JNK inhibitor, but not p38 MAPK inhibitors, significantly decreased the UVA-induced expression of uPA by pterygium fibroblasts. Normal conjunctival fibroblasts were less sensitive to UVA irradiation compared to the pterygium fibroblasts. CONCLUSIONS: UVA stimulated the production of uPA, a key factor in the modulation of extracellular matrixes, inflammatory processes, and angiogenesis. This may have a role in the development and progression of pterygium.

Immunohistochemical analysis of angiotensin converting enzyme in Sardinian pterygium.

Pterygium is a common ocular surface disorder characterized by excessive cell proliferation, inflammation, fibrosis, angiogenesis and extracellular matrix remodeling. The Angiotensin converting enzyme (ACE or ACE I) is the major component of the Renin-angiotensin system (RAS) converting the inactive decapeptide Angiotensin I (Ang I) to the active octapeptide Angiotensin II (Ang II). Besides this 'classical role', it can act as transcriptional regulator in response to external stimuli that may lead to cell damage and tissue remodeling. Due to this role, it can be internalized into the nuclear compartment to act as transcriptional factor for proteins involved in the inflammatory response. The aim of the present study was to determine ACE expression and localization in pterygium and culture pterygium cells by immunohistochemistry. Our results are the first to demonstrate nuclear immunolocalization of ACE, more so in pterygium compared to conjunctiva epithelial cells in histological sections. ACE was not detected in the nuclei of subcultivated pterygium epithelial cells. The nuclear localization of ACE may be correlated with an anti-inflammatory path mediated by activation of its transcriptional role.

Activation of MAPK signaling pathway and NF-kappaB activation in pterygium and ipsilateral pterygium-free conjunctival specimens.

PURPOSE: To evaluate mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways in pterygium and pterygium-free conjunctivas. METHODS: Primary pterygia (n = 21), ipsilateral superior-temporal bulbar conjunctivas (n = 8), and healthy conjunctival (n = 5) biopsy specimens were analyzed. Total and phosphorylated (phospho) levels of extracellular-regulated 1/2 (ERK1/2), p38, and c-jun N-terminal (JNK) MAPKs and NF-κB inhibitor-alpha (IκΒ-α) were analyzed by immunobead-based assay. Tissue phospho-, total protein, and activation values determined by phospho/total ratios were compared. Correlation among those values and clinical parameters were determined. Average-linkage hierarchical cluster analysis identified patients with similar protein activation values. The k-nearest neighbor classifier predicted the origin of specimens based on protein levels. RESULTS: Pterygium samples had significantly lower total JNK and IκΒ-α levels than did healthy conjunctivas. Decreased total JNK and IκΒ-α and increased phospho-IκΒ-α levels and phospho/total ratio of JNK and IκΒ-α were present in ipsilateral conjunctivas compared with healthy conjunctivas. Protein levels were correlated among them in pterygium, ipsilateral, and healthy conjunctivas and with sun exposure, pterygium grade, and pterygium measurements. Cluster analysis of activation values and ratios in pterygium and ipsilateral-conjunctiva revealed different groups of patients with similar values. Prediction accuracy was 70% to 80% for the classifiers phospho- and total protein levels and phospho/total ratio. CONCLUSIONS: Pterygium and pterygium-free ipsilateral conjunctivas had alterations in MAPK and NF-κB pathways not present in healthy conjunctivas. The high prediction accuracy based on phospho- and total protein levels and phospho/total ratio of ERK1/2, p38, JNK, and IκB-α suggests these molecules as potential biomarkers of inflammation in pterygia.

Study of alkaline phosphatase activity and DNA content of pterygium tissue showing its degenerative character.

A prospective, non-randomised, interventional case series study was designed among 127 specimens obtained from 100 consecutive patients presenting with primary pterygium attending the outpatient department of a teaching hospital in Kolkata, West Bengal between September 2009 and August 2010. After harvesting the pterygium it was transported in 10% formol saline for staining, fixing and embedding prior to evaluation of tissue alkaline phosphatase level and estimation of DNA content in nucleus of cells in pterygium tissue. The main outcome was to measure alkaline phosphatase activity and DNA content in pterygium tissue and normal bulbar conjunctiva along with histopathological staining and to see if any correlation exists between alkaline phosphatase activity and DNA content of pterygium tissue in different stages of evolution as suggested by histological staining. The elastic proliferation and hyaline degeneration are prominent in well developed pterygium. The alkaline phosphatase activity in all cases of pterygium is maximum and the DNA content of the cells of the pterygium tissue is diminished as compared to normal bulbar conjunctiva. Histopathological changes as evident from epithelial cell changes from cylindrical to flat cells with cyst formation in basal layers, the disappearance of nuclei in the submucosa coupled with increase in hyaline content of the tissue all point towards degenerative changes. Biochemically too there is a decrease in DNA content in the nuclei of the cells in pterygium, histochemically there is an increase in the alkaline phosphatase activity, all these changes point to pterygium being a degenerative tissue.

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.

Effect of TIMP-1 and MMP in pterygium invasion.

PURPOSE. The migration and invasion of tumor cells correlate with the interaction between MMP and TIMP. Therefore, the purpose of this study was to determine the role of MMP-9, MMP-10, and TIMPs in pterygium formation and progression. METHODS. MMP-9, MMP-10, and TIMP proteins were studied using immunohistochemistry on 82 pterygial specimens and 30 normal conjunctivas. Pterygium epithelial cells (PECs), cultured in a serum-free culture medium, and siRNA were used to knock down TIMP gene expression to understand the role of TIMP in pterygium invasion. RESULTS. Among the 82 pterygial samples, 29 specimens (35.4%) were positive for MMP-9 expression, 28 were positive for MMP-10 (34.1%), and 59 were positive for TIMP1 (72.0%). Staining for MMPs was limited to the cytoplasm of the epithelial layer. The TIMP staining was detected in the pterygium epithelium, fibroblasts and corneal epithelium. In the cell model, cell invasion and migration ability increased in TIMP knockdown PECs compared with the parental control. CONCLUSIONS. MMP-9 and MMP-10 may each play a role in pterygium formation, and TIMPs may contribute to pterygium invasion inhibition.

Increased expression of gelatinase (MMP-2 and MMP-9) in pterygia and pterygium fibroblasts with disease progression and activation of protein kinase C.

PURPOSE: To study the expression of matrix metalloprotease (MMP)-2 and MMP-9 mRNA and activities in various stages of surgically excised pterygium specimens and cultured pterygium fibroblasts and to study the effects of activation of protein kinase C (PKC) on the expression of these MMPs in pterygium fibroblasts. METHODS: MMP-2 and MMP-9 mRNA expression and activities in 15 pterygium tissues and cultured pterygium fibroblasts were measured by RT-PCR and zymography. Five normal conjunctiva specimens and fibroblasts were tested as the controls. Changes of expression of MMP-2 and MMP-9 of fibroblasts after the simulation of a standard PKC activator, 2-O-tetradecanoyl-phorbol-13-acetate (TPA), were studied. RESULTS: MMP-2 and MMP-9 expression in pterygium tissues and fibroblasts was greater than those of normal tissues and fibroblasts and was closely relevant to the progression of pterygium. In early-stage pterygium tissues and cultured fibroblasts, MMP-9 was not expressed, activated MMP-2 could not be detected, and only a small amount of latent MMP-2 was present. In advanced-stage pterygium (pterygium head passed the papillary region), MMP-9 was expressed; activated MMP-2 and a large amount of latent MMP-2 could be detected in pterygium tissues and fibroblasts. TPA stimulated the expression of MMP-2 and MMP-9 by pterygium fibroblasts isolated from early-stage specimens in a dose-dependent manner. CONCLUSIONS: MMP-2 and MMP-9 expression by pterygium fibroblasts is significantly increased after the progression of pterygium. Activation of the PKC signaling pathway, aside from other previously reported signaling pathways, may play a role in the development and progression of 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.

[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.

Phospholipase D in the human ocular surface and in pterygium.

PURPOSE: Pterygium is a fibro-vascular disease of unknown etiology characterized by proliferation and advancement of tissue onto the cornea. Phospholipase Ds (PLDs) are members of an important class of enzymes involved in inflammation and differentiation. In cultured corneal epithelial cells, these enzymes play a role in wound healing, and in other contexts, they suppress apoptosis and increase cell motility. We aimed to study the presence of PLD subtypes in native ocular surface tissue and pterygium. METHODS: This study involved paired control or uninvolved conjunctival and pterygium tissues from 6 patients. Reverse transcription semiquantitative and quantitative polymerase chain reactions were performed to assess transcript levels for PLD1-5 in normal conjunctiva and pterygium tissue. Immunofluorescent staining by using antibodies against PLD1/2 was used to study the expression and tissue distribution. Western blots were performed for protein detection and to confirm the specificity of the antibodies used. RESULTS: PLD1, 2, 3, and 4 transcripts were detected in normal conjunctiva tissue, and types 2, 3, and 4 were upregulated in pterygium. Immunofluorescent staining showed the presence of phospholipase-D1/2 in normal cornea, conjunctival, and pterygial epithelia. In normal cornea and conjunctival epithelia, the expression was mainly localized to the nuclei of the basal and suprabasal epithelial cells, whereas in pterygium, this expression was limited to the cytoplasm and peri-plasma membrane regions. Western blot confirmed the presence of PLD1/2 in proteins extracted from pterygium and conjunctiva tissue. CONCLUSIONS: PLD subtypes are present in human ocular surface epithelium. PLD may be involved in pterygium pathogenesis.

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.