Original Article

Cyclooxygenase-2 expression in primary and recurrent pterygium

Nermin Karahan, MD; Sirin Baspinar, MD; Metin Ciris, MD; Cetin LutÞ Baydar, MD; Nilgun Kapucuoglu, MD

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 inßammation. 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
signiÞcant difference in COX-2 expressions in Þbroblasts between primary and recurrent pterygium cases
(P = 0.001). There were statistically signiÞcant differences in COX-2 expressions in surface epithelium
(P = 0.028) and stromal inßammatory cells (P=0.000) between control tissues and primary pterygia tissues.
We also detected statistically signiÞcant differences in COX-2 expressions in surface epithelium (P=0.000),
stromal Þbroblasts P=0.000 (stromal Þbroblasts and inßammatory cells), vessels (P = 0.027) and inßammatory
cells (P=0.001) between control tissues and recurrent pterygia tissues.

Conclusions: This is the Þrst study to document the expression of COX-2 in primary and recurrent pterygia. In
our opinion after excision of pterygia, Þbroblastic proliferation continues and this contributes to recurrence.

Key words: Cyclooxygenase-2, immunohistochemistry, primary pterygium, recurrent pterygium

Indian J Ophthalmol 2008;56:279-83

Pterygia are common, frequently recurring ocular surface lesions Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)
characterized by tissue remodeling, cellular proliferation, mediate the rate-limiting step in arachidonic acid metabolism.11
angiogenesis, and inßammation.1,2 Irritation of the eye by Expression of COX-2 mRNA and protein is often enhanced in
ultraviolet (UV) light in sunny, dry, dusty areas and repeated various human cell types by inßammatory cytokines such as
microtrauma can lead to development of pterygium in interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).11
susceptible individuals.2 Pterygia are histopathologically Both isoforms of cyclooxygenase, constitutive COX-1 and
Þbrovascular proliferations. This expansiveness of Þbrovascular inducible COX-2, catalyze the production of prostanoids from
proliferation is considered as a convenient morphological index arachidonic acid.11 COX-2-induced production of prostanoids
for evaluation of recurrence risk after surgical treatment.1-3 is often implicated in inßammatory diseases, characterized
Pterygia usually recur after surgical excision.4 The cause by edema and tissue injury due to the release of many
of recurrence may be related to several factors including inßammatory cytokines and chemotactic factors, prostanoids,
environmental factors. If etiological and pathogenic differences leukotrienes, and phospholipase.11,12
between recurrent and non-recurrent cases are discovered, the
type of treatment can be more easily selected and convenient COX-2 expression is induced by various stimuli, and the
treatment can be found.1-6 over-expression is closely related to the pathogenesis of some
degenerative diseases including cancer.13 Several lines of
Histopathological Þndings reveal epithelial hyperplasia evidence indicate that COX-2 over-expression can be a causal
and exuberant Þbrovascular tissue in the stroma of pterygia.6 factor for tumor growth and metastasis. Over-expression of
Importantly, the extent of Þbrovascular proliferation in the COX-2 in vitro promotes cell proliferation in human prostate
stroma has been used as a reliable morphological index for cancer cells and enhances invasiveness of human bladder,
predicting pterygium recurrence following primary excision.7-10 breast and colon cancer cells.14-17 Moreover, the findings
regarding the effects of COX-2 in cutaneous tumor formation
Departments of Pathology (NK, MC, NK), Forensic Medicine (CLB), have also been reported in pterygium, including disruption
Suleyman Demirel University School of Medicine, Isparta, Turkey, of apoptosis, limbal epithelial proliferation, abnormal p53
Department of Pathology (SB), Egirdir Bone Joint Diseases Treatment gene expression, and upregulation of basic Þbroblast growth
and Rehabilitation Hospital, Isparta, Turkey factor, vascular endothelial growth factor, and nitric oxide
Correspondence to Dr. Nermin Karahan, Suleyman Demirel University,
synthase.8,18-21
School of Medicine, Department of Pathology, Isparta-Turkey. The aim of the study was to find out the relationship
E-mail: nerminkarahan@hotmail.com between COX-2 expression and primary as well as recurrent
Manuscript received: 17.09.07; Revision accepted: 10.01.08 pterygium.
280 Indian Journal of Ophthalmology Vol. 56 No. 4

Materials and Methods Results

Resected pterygium tissue samples from 33 patients of 21 Our group consisted of 23 (59%) men and 16 (41%) women.
with primary pterygium, 12 with recurrent pterygium and Ages of the patients ranged from 36 to 80 (median 54 years).
six normal corneo-scleral tissues were included in this study. Eleven of 21 primary pterygium cases were males and 10 of
Primary and recurrent pterygium samples were selected them were females. Eight of the recurrent pterygium cases were
retrospectively from archival material of the department of males and four were females. Four of the six healthy conjunctiva
pathology, Suleyman Demirel University School of Medicine. cases were males and two of them were females.
All pterygia were sectioned along the longitudinal axis to
COX-2 immunostaining was observed mainly in the
include from the cap (leading edge) to the basal (body) region.
cytoplasm of surface epithelium, stromal inßammatory cells,
Normal corneo-scleral tissues were obtained during forensic
capillary vessels and fibroblasts. There were statistically
autopsy cases with attorney’s permission.
signiÞcant differences in COX-2 expressions in the surface
Immunohistochemical analysis for COX-2 was performed epithelium (P = 0.028) and stromal inßammatory cells (P=0.000)
on formalin-Þxed, paraffin-embedded archival tissue using between control tissues and primary pterygia tissues.
the streptavidin-biotin-peroxidase technique. For all cases,
We also detected statistically signiÞcant differences in COX-2
4 µm histological section was deparaffinized in xylene and
expressions in surface epithelium (P=0.000), stromal Þbroblasts
dehydrated in descending dilution of ethanol. For the antigen
P=0.000 (stromal Þbroblasts and inßammatory cells), vessels
retrieval, slides were treated by microwave heating in citrate
(P = 0.027) and inßammatory cells (P=0.001) between control
buffer (pH 6.0) for 10 min. Endogenous peroxidase activity
tissues and recurrent pterygia tissues.
was blocked by 20 min of incubation with 0.3% hydrogen
peroxidase. Slides were tested with COX-2 antibody (1:100 Statistical significance was not observed in epithelial
Epitope speciÞc rabbit antibody, Lab Vision). Sections were staining of primary and recurrent pterygium cases (P = 0.06).
tested with streptavidin-biotin-peroxidase kit (Ultra Vision In 14 primary pterygium cases and in all recurrent pterygium
Large Volume Detection System Anti-polyvalent, HRP, Lab cases, COX-2 expressions were detected in fibroblasts. In
Vision, USA), and after incubation the reaction product all the recurrent pterygium cases COX-2 showed strong
was detected using diaminobenzidine (DAB). Finally, the expression. There was a statistically signiÞcant difference in
sections were counterstained with Mayer’s hematoxylin, and COX-2 expression in Þbroblasts between primary and recurrent
mounted with mounting medium. Tissue of colon cancer pterygium cases (P = 0.001).
from a human served as the positive control in the COX-2
immunostaining. COX-2 expressions in both epithelium and stroma of normal
corneo-scleral tissue, primary and recurrent pterygia are
Two observers analyzed the staining for COX-2. shown in Figs. 1-3. The relation between COX-2 expressions in
Immunohistochemical analysis of the primary and recurrent epithelium and stroma of normal corneo-scleral tissue, primary
pterygium was performed with anti-COX-2 antibody. The and recurrent pterygia are shown in Tables 1-3.
mean overall intensity of the immunostaining in the surface
epithelium and stromal cells was scored in the analyzed Discussion
sections of primary and recurrent pterygium samples as Pterygium has long been considered to be a chronic degenerative
follows: 0 = absent immunostaining; 1 = weak immunostaining condition. However, after over-expression of the p53 protein
(few cells being positive focal or scattered); 2 = strong was found in the epithelium of pterygium, some researchers
immunostaining (diffuse staining throughout the tissue). The began to feel that pterygium was an ultraviolet (UV)-related
histology was checked using slides stained with Hematoxylin tumor rather than a degenerative disease.18,19
Eosin.
Although the pathogenesis of pterygia is still poorly
For statistical evaluation, the SPSS software version 13.0 was understood, epidemiologic evidence suggests that environmental
used. The Fisher exact and chi-square tests were used to analyze stress may have a role.1,3 Recently, several cytokines such as
the distribution of COX -2 positive cases according to several TNF-α, Þbroblast growth factor (FGF), and transforming growth
histopathological features. P-value < 0.05 was considered as factor-β (TGF-β), have been localized to both resident and
signiÞcant. inßammatory cells in pterygia.2,8 Kria et al.,8 recently reported

Table 1: Cyclooxygenase-2 expressions in epithelium and stroma of normal corneo-scleral tissue and primary pterygia
Groups Cyclooxygenase-2 expression
Normal corneo-scleral tissue Primary pterygia tissue P-value
Negative Weak Strong Negative Weak Strong
Epithelium 0 6 (100) 0 2 (9.5) 8 (31.8) 11 (52.4) 0.028*
Stroma
Fibroblast 2 (33.3) 4 (66.7) 0 7 (33.3) 7 (33.3) 7 (33.3) 0.195*
Inflammatory cells 6 (100) 0 0 3 (14.3) 6 (28.6) 12 (57.1) 0.000*
Capillary vessels 0 6 (100) 0 4 (19) 12 (51.7) 5 (23.8) 0.145*
*Pearson Chi-square. Figures in parentheses are in percentage
July - August 2008 Karahan et al.: COX-2 expression in primary and recurrent pterygium 281

Figure 1: Weak COX-2 expression in epithelium and stroma of normal corneo-scleral tissue (A) (COX-2X100), (B) (COX-2X200)

Figure 2: Moderate COX-2 expression in epithelium and stroma of primary pterygium tissue (A) (COX-2X100), (B) (COX-2X200)

Figure 3: Strong COX-2 expression in epithelium and stroma of recurrent pterygium tissue (A) (COX-2X100), (B) (COX-2X200) Epithelium (E),
fibroblasts (F), capillaries (C) and inflammatory cells (I) were shown in figures with arrows
that pterygium Þbroblasts express potent Þbroangiogenic factors COX-2 is an inducible immediate-early gene which is up-
such as basic Þbroblast growth factor (b-FGF), platelet derived regulated by various stimuli, including mitogens, cytokines,
growth factor (PDGF), TGF-β and TNF-α, suggesting that they growth factors, and tumor promoters.11 Extensive evidence
may have a possible role in pterygium pathogenesis. indicates that COX-2 prostanoid pathway is involved in
282 Indian Journal of Ophthalmology Vol. 56 No. 4

Table 2: Cyclooxygenase-2 expressions in epithelium and stroma of normal corneo-scleral tissue and recurrent pterygia
Groups Cyclooxygenase-2 expression
Normal corneo-scleral tissue Recurrent pterygia tissue P-value
Negative Weak Strong Negative Weak Strong
Epithelium 0 6 (100) 0 0 1 (8.3) 11 (92.7) 0.000**
Stroma
Fibroblast 2 (33.3) 4 (66.7) 0 0 0 12 (100) 0.000*
Inflammatory cells 6 (100) 0 0 0 3 (25) 9 (75) 0.000*
Capillary vessels 0 6 (100) 0 1 (8.3) 4 (33.3) 7 (58.3) 0.027*
*Pearson Chi-square’; **Fisher’s exact test. Figures in parentheses are in percentage

Table 3: Cyclooxygenase-2 expressions in epithelium and stroma of primary and recurrent pterygia
Groups Cyclooxygenase-2 expression
Primary pterygia tissue Recurrent pterygia tissue P-value
Negative Weak Strong Negative Weak Strong
Epithelium 2 (9.5) 8 (31.8) 11 (52.4) 0 1 (8.3) 11 (92.7) 0.068*
Stroma
Fibroblast 7 (33.3) 7 (33.3) 7 (33.3) 0 0 12 (100) 0.001*
Inflammatory cells 3 (14.3) 6 (28.6) 12 (57.1) 0 3 (25) 9 (75) 0.344*
Capillary vessels 4 (19) 12 (51.7) 5 (23.8) 1 (8.3) 4 (33.3) 7 (58.3) 0.137*
*Pearson Chi-square. Figures in parentheses are in percentage

inßammation.12 COX-2 modulates angiogenesis by increasing It is not yet clear how the tissue alterations are initiated and
the production of angiogenic factors, such as vascular kept in progress.25,26 Controversial factors include the mutation
endothelial growth factor.11,12 Chiang et al.,22 investigated of limbus stem cells, modification of stromal fibroblasts,
the expression of COX-2 in pterygium and found 75 (83.3%) and immunological and other processes in the matrix. In
specimens stained positive for COX-2 in the pterygium new models of pterygial pathogenesis, interactions between
group. The staining was limited to the cytoplasm of the epithelial cells, Þbroblasts, and matrix are considered as well
epithelial layer and predominantly over the basal epithelial as the denaturing action of the matrix metalloproteinases.27
layer. No substantial staining was visible in the subepithelial
The management of pterygia is still a challenge for the
Þbrovascular layers. All specimens were negative in the normal
ophthalmologist. Many surgical procedures have been
conjunctiva and limbus group. In our study we detected COX-2
proposed for pterygium removal.28 However, surgery alone
expression both in normal corneo-scleral tissues, primary
is not sufficient and cannot prevent a high incidence of
pterygia and recurrent pterygia in superÞcial epithelium and
recurrence.28 There are theories that the causes considered
also in stromal cells with immunohistochemical methods. We
as etiological agents in recurrence development in pterygia
also found that COX-2 expressions were signiÞcantly more
are inßammations besides UV-rays and other environmental
intense in stromal Þbroblasts of recurrent pterygia compared
factors.2 In our opinion after excision of pterygia, Þbroblastic
to primary pterygia. This Þnding manifests that Þbroblastic
proliferation continues and this contributes to recurrence.
proliferation is more developed in recurrent pterygia compared
to primary pterygia. However if a Þbroblastic cell invasion of the cornea is
considered to be the essential pathology, then a recurrence could
Also, in all the recurrent pterygia there were COX-2
be ascribed to the same cause, namely, an accelerated reinvasion
expressions detected in stromal inßammatory cells. Although
of the cornea. As Cameron29 indicated, the recurrence would
we observed that COX-2 expression is more excessive in
appear to be due to an accelerated Þbroblastic proliferation
recurrent pterygia compared to primary pterygia, this was
produced by the trauma of operation. In our opinion
not statistically signiÞcant. It may be due to our small series
Þbroblastic proliferation explains the clinical appearance and
of recurrent pterygia.
behavior of a recurrent pterygium. As a result, in the light of
Pterygium is now considered to be a result of uncontrolled these Þndings, we think that after pterygium excision, selective
cellular proliferation, similar to that in tumors, in which there COX-2 inhibitors may be helpful to prevent recurrence.
is damage to cellular regulation and control of the cell cycle.23
Mitogenity, construction of a new vascular net and remodeling References
of the extracellular matrix were observed in pterygia. Altogether
1. Coroneo MT, Girolamo N, WakeÞeld D. The pathogenesis of
they create a new vascular and Þbrotic tissue, which has an
pterygia. Curr Opin Ophthalmol 1999;10:282-8.
aggressive way of growing to and over the cornea.24
2. Hill JC, Maske R. Pathogenesis of pterygium. Eye 1989;3:218-26.
Changes in the proteoglycans are known to induce 3. Degrassi M, Piantanida A, Nucci P. Unexpected histological
edematous changes, which in turn cause Þbroblast proliferation. Þndings in pterygium. Optom Vis Sci 1993;70:1058-60.
July - August 2008 Karahan et al.: COX-2 expression in primary and recurrent pterygium 283

4. Avisar R, Arnon A, Avisar E. Primary pterygium recurrence time. 2002;299:886-90.

Isr Med Assoc J 2001;3:836-7. 17. Singh B, Berry JA, Shoher A, Ramakrishnan V, Lucci A. COX-2
5. Marvovich A, Morad Y, Sandbank J. Angiogenesis in pterygium: overexpression increases motility and invasion of breast cancer
Morphometric and immunohistochemical study. Curr Eye Res cells. Int J Oncol 2005;26:1393-9.
2002;25:17-22. 18. Tan DT, Lim AS, Goh HS, Smith DR. Abnormal expression of the
6. Tan D, Chee SP, Dear K, Lim AS. Effect of pterygium morphology on p53 tumor suppressor gene in the conjunctiva of patients with
pterygium recurrence in a controlled trial comparing conjunctival pterygium. Am J Ophthalmol 1997;123:404-5.
autografting with bare sclera excision. Arch Ophthalmol 19. Tan DT, Tang WY, Liu YP, Goh HS, Smith DR. Apoptosis and
1997;115:1235-40. apoptosis related gene expression in normal conjunctiva and
7. Touhami A, Di Pascuale MA, Kawatika T, Del Valle M, Rosa RH Jr, pterygium. Br J Ophthalmol 2000;84:212-6.
Dubovy S et al. Characterisation of myoÞbroblasts in Þbrovascular 20. Dushku N, John MK, Schultz GS, Reid TW. Pterygia pathogenesis:
tissues of primary and recurrent pterygia. Br J Ophthalmol Corneal invasion by matrix metalloproteinase expressing
2005;89:269-74. altered limbal epithelial basal cells. Arch Ophthalmol 2001;119:
8. Kria L, Ohira A, Amemiya T. Immunohistochemical localization 695-706.
of basic Þbroblast growth factor, platelet derived growth factor, 21. Lee DH, Cho HJ, Kim JT, Choi JS, Joo CK. Expression of vascular
transforming growth factor and tumor necrosis factor in a primary endothelial growth factor and inducible nitric oxide synthase in
pterygium. Acta Histochem 1996;98:195-201. pterygia. Cornea 2001;20:738-42.
9. Di Girolamo N, Coreneo MT, WakeÞeld D. UVB-Elicited induction 22. Chiang CC, Cheng YW, Lin CL, Lee H, Tsai FJ, Tseng SH,
of MMP-1 expression in human ocular surface epithelial cells is Tsai YY. Cyclooxygenase 2 expression in pterygium. Mol Vis
mediated through the ERK1/2 MAPK- dependent pathway. Invest 2007;13:635-8.
Ophthalmol Vis Sci 2003;44:4705-14.
23. Perra MT, Maxia C, Corbu A, Minerba L, Demurtas P, Colombari R,
10. Marcovich A, Morad Y, Sandbank J. Angiogenesis in pterygium: et al. Oxidative stress in pterygium: Relationship between p53 and
Morphometric and immunohistochemical study. Curr Eye Res 8-hydroxydeoxyguanosine. Mol Vis 2006:12; 1136-42.
2002;25:17-22.
24. Solomon AS. Pterygium. Br J Ophthalmol 2006;90:665-6.
11. Williams CS, Mann M, DuBois RN. The role of cyclooxyigenases
in inflammation, cancer and development. Oncogene 25. Sakoonwatanyoo P, Tan DT, Smith DR. Expression of p63 in
1999;18:7908-16. pterygium and normal conjunctiva. Cornea 2004;23:67-70.

12. Kuwano T, Nakao S, Yamamoto H, Tsuneyoshi M. Cyclooxygenase 26. Saw SM, Tan D. Pterygium: Prevalence, demography and risk
2 is a key enzyme for inßammatory cytokine-induced angiogenesis. factors. Ophthalmic Epidemiol 1999;6:219-28.
FASEB J 2004;18:300-10. 27. Seifert P, Eckert J, Spitznas M. Topological-histological
13. Smith WL, Garavito RM, DeWitt DL. Prostaglandin endoperoxide investigation of the pterygium. Graefes Arch Clin Exp Ophthalmol
H synthases (cyclooxygenases)-1 and -2. J Biol Chem 2001;239:288-93.
1996;271:33157-60. 28. Hirst LW. Treatment of pterygium. Aust N Z J Ophthalmol
14. Tsujii M, Kawano S, DuBois RN. Cyclooxygenase-2 expression in 1998;26:269-70.
human colon cancer cells increases metastatic potential. Proc Natl 29. Cameron ME. Histology of pterygium: An electron microscopic
Acad Sci USA 1997;94:3336-40. study. Br J Ophthalmol 1983;67:604-8.
15. Fujita H, Koshida K, Keller ET, Takahashi Y, Yoshimito T, Namiki M,
et al. Cyclooxygenase-2 promotes prostate cancer progression.
Prostate 2002;53:232-40.
16. Li G, Yang T, Yan J. Cyclooxygenase-2 increased the angiogenic and
Source of Support: Nil, Conflict of Interest: None declared.
metastatic potential of tumor cells. Biochem Biophys Res Commun

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