laboratory sciences pterygia pathogenesis

Pterygia Pathogenesis

Corneal Invasion by Matrix MetalloproteinaseExpressing Altered Limbal Epithelial Basal Cells

Nicholas Dushku, MD; Molykutty K. John, PhD; Gregory S. Schultz, PhD; Ted W. Reid, PhD

Objective: To assess the potential role of matrix metal-loproteinases (MMPs) in the pathogenesis of pterygia bycomparing the immunolocalization patterns of MMPs inaltered limbal basal stem cells, activated fibroblasts, andareas of elastotic degeneration adjacent to the pterygia.

Methods: Nine primary and 1 recurrent pterygia alongwith normal superior limbal-conjunctival tissue and cor-nea were immunostained with mouse monoclonal anti-bodies specific for MMP-1, MMP-2, MMP-3, MMP-9,membrane type 1 (MT1)–MMP (MMP-14), and mem-brane type 2–MMP (MMP-15).

Results: Normal conjunctival, limbal, and corneal cellslacked significant immunostaining except for cell surfaceMT1-MMP. In contrast, altered limbal basal epithelial cellsof the 9 primary and 1 recurrent pterygia immunostainedfor all 6 MMPs. Activated and altered fibroblasts associ-ated with the pterygia immunostained primarily for MMP-1.In contrast, stromal areas of elastotic degeneration (pin-gueculae) showed variable immunostaining of MMPs.

Conclusions: Altered limbal basal epithelial cells (pte-rygium cells) immunostained for multiple types ofMMPs in contrast to normal conjunctival, limbal, andcorneal cells. The pterygium cells invading over Bow-man’s layer produce elevated MMP-1, MMP-2, andMMP-9 expression, which probably are the main MMPsresponsible for the dissolution of Bowman’s layer. Pte-rygium cells may also cause activation of fibroblasts atthe head of the pterygium, leading to the initial cleavageof fibrillar collagen in Bowman’s layer by the produc-tion of MMP-1. Altered fibroblasts in areas of elastoticdegeneration (pingueculae) trailing behind the pte-rygium constitute a second type of tumor, which isnoninvasive.

Clinical Relevance: These data of altered MMP ex-pression support the concept that altered basal limbal epi-thelial cells play a key role in the formation and migra-tion of a pterygium.

Arch Ophthalmol. 2001;119:695-706

I N OUR SEARCH for the pathogen-esis of pterygia, several importantclinical and pathologic character-istics of primary and recurrent pte-rygia have emerged:

1. Epidemiological studies havefirmly established that UV-B radiation cor-relates as the etiologic agent for pte-rygia1,2 and limbal tumors.3-5

2. Pterygia begin growing from lim-bal epithelium and not from conjunctivalepithelium.6,7

3. A segment of limbal epithelium,the migrating limbus, invades the corneacentripetally followed by conjunctival epi-thelium.8-10

4. A distinct type of corneal cells de-velops at the leading edge of the pterygiatissue.9,11,12

5. Vascularization occurs in the con-junctiva adjacent to pterygia.13

6. Bowman’s layer is dissolved un-der the leading edge of the pterygia.14

7. Pterygia have a high recurrencerate.11,12

Since UV-B is known to be muta-genic for the TP53 tumor suppressorgene,15-18 we previously searched for ab-normal TP53 expression in pterygia, lim-bal tumors, and pingueculae from whichthese 2 growths seem to originate.19 Wefound nuclear p53 expression withoutapoptosis in the limbal epithelia of pte-rygia, limbal tumors, and most pinguecu-lae. This suggested to us that mutation inTP53 or mutations in the p53 pathway forapoptosis may occur as an early event inthe tumorlike development in these cells,which is consistent with their causationby UV radiation. As a consequence of mu-tational damage to the p53-dependent pro-grammed cell death mechanism,20 muta-tions in other genes could progressively beacquired by the altered limbal basal epi-thelial cells. This is consistent with the con-cept of a multistep21 development of tu-morlike pterygium cells that arise fromaltered limbal epithelial cells overlying apinguecula. We also discovered that pri-mary and recurrent pterygia were charac-

LABORATORY SCIENCES

From the Department ofOphthalmology, KaiserPermanente Medical Center,Sacramento, Calif(Dr Dushku); Department ofObstetrics and Gynecology,Institute for Wound Healing,University of Florida,Gainesville (Drs John andSchultz); and Departments ofOphthalmology and VisualSciences, Texas TechUniversity, Lubbock (Dr Reid).

(REPRINTED) ARCH OPHTHALMOL / VOL 119, MAY 2001 WWW.ARCHOPHTHALMOL.COM695

©2001 American Medical Association. All rights reserved.Downloaded From: https://jamanetwork.com/ on 05/08/2022

terized by invasion of the cornea by vimentin-expressingaltered limbal epithelial basal cells.8,9 In addition, we dis-covered there is local infiltration of the adjacent conjunc-tival and circumferential limbal epithelia by pterygium cells,which could lead to a high recurrence rate if not con-trolled surgically or chemotherapeutically.9 Moreover, us-ing polymerase chain reaction, we found no human pap-illomavirus DNA in any of these growths that arise in theUV-exposed interpalpebral region. We concluded that hu-man papillomavirus DNA is not required as a cofactor forthe etiology of these lesions, either through control of theaction of p53 or through any other mechanism.22,23

These data have led us to propose an integratedmodel for the formation and pathophysiology of pte-rygia and pinguecula.9,19,24 A key component of this hy-pothesis is that the true pterygium cells are tumorlike al-tered limbal epithelial basal cells that have altered TP53tumor suppressor gene expression. With accumulationof sufficient mutations, the pterygium cells invade ontonormal corneal basement membrane and draw conjunc-tival epithelial cells along with them.

If this hypothesis is correct, we would predict thatthe expression of proteases that degrade basement mem-

brane components, such as type IV collagen and the fi-brillar collagen of corneal stroma, should be elevated inthe leading edges of pterygia, where the degradation of Bow-man’s layer occurs. Also, the normal limbal and conjunc-tival epithelia and stroma should lack these proteases (orhave low levels of the proteases). The primary class of pro-teases that degrade matrix are the matrix metalloprotein-ases (MMPs). The MMPs are a family of more than 21 ge-netically distinct proteases, which are normally producedin small amounts for physiological processes by cells, suchas fibroblasts and epithelial cells.25 Recently, it was re-ported that fibroblasts from pterygia when grown in cul-ture exhibited elevated MMP expression.26,27 In general,invasive tumor cells are known to overexpress MMPs28,29

of various types depending on the tumor.30-34 These pro-teases released by tumor cells facilitate invasion by de-grading components of their basement membranes and ad-jacent stromal matrix. Previously, we proposed a modelfor pterygia migration and dissolution of Bowman’s layerinvolving proteases.19,24 Recently, elevated expression ofMMPs was demonstrated in pterygia.35,36 Specific local-ization of MMPs in the altered limbal epithelial basal cellsof pterygia had not been reported previously. In this ar-

MATERIALS AND METHODS

PTERYGIA AND NORMAL TISSUE

In compliance with the World Medical Association Decla-ration of Helsinki, 9 primary and 1 recurrent pterygia weresurgically removed in the ambulatory surgery center at theKaiser Permanente Medical Center, Rancho Cordova, Calif,and processed as described previously.9,19,23 Briefly, to iden-tify the invading limbus epithelium with altered limbal basalcells and the zone of dissolution of Bowman’s layer, inci-sions were made in the cornea 1 to 2 mm central to the lead-ing edge of the pterygium and deep enough to include Bow-man’s layer. The incisions were extended into the adjacentconjunctiva for 5 to 6 mm posterior to the surgical limbusand 1 to 2 mm beyond the superior and inferior conjunc-tival folds. For proper orientation, specimens were su-tured onto sterile cardboard and immediately fixed in 10%neutral buffered formalin for 6 to 10 hours, then embed-ded in paraffin. Serial cross sections of pterygia specimenswere made along the longitudinal axis to include the lead-ing edge of cornea-invading altered limbal basal cells overBowman’s layer, the migrating limbus, and adjacent con-junctiva. Every tenth section was stained with hematoxylin-eosin to locate these 3 areas. For immunostaining, sec-tions were selected that contained cornea-type cells betweenthe dissolved edge of Bowman’s layer and conjunctiva (asindicated by the presence of goblet cells). A specimen offresh normal human superior limbal-conjunctival tissueserved as a normal tissue control. In addition, sections ofnormal cornea (obtained along with the pterygia) also servedas internal controls. Three cadaver eyes placed in 10% neu-tral buffered formalin 4 to 5 hours after death were usedfor comparison with the fresh surgical specimens (Table1,only 5-hour specimen results are shown). Human pla-centa, which is known to produce MMPs, was used as apositive tissue control.38 Additional negative controls used

pterygia tissues incubated without the primary antibodiesto MMPs.

IMMUNOHISTOCHEMISTRY

Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded tissue sections using the avidin-biotin-peroxidase complex method as described previ-ously.39 Briefly, sections 5 µm thick were cut anddeparaffinized in xylene and descending ethanol series. En-dogenous peroxidase activity was destroyed by a 20-minute treatment at room temperature with 3% hydrogenperoxide in distilled water. Sections were then incubatedfor 1 hour at room temperature in a humidified chamberwith primary mouse monoclonal antibodies directed againstthe MMPs. The following mouse monoclonal antibodieswere used: MMP-1, MMP-2, MMP-3, and MMP-9, whichwere all diluted 50-fold (Oncogene Research Products, Bos-ton, Mass), and membrane type 1 (MT1)–MMP and mem-brane type 2 (MT2)–MMP, which were diluted 100-fold(Chemicon International Inc, Temecula, Calif). Sectionswere washed and then incubated with a biotinylated sec-ondary antibody directed against the mouse monoclonalantibodies for 1 hour at room temperature in a humidifiedchamber using the Dako LSAB Kit (Dako Corporation,Carpinteria, Calif). Sections were washed and then incu-bated with 0.05% 3,39-diamino-benzidine tetrahydrochlo-ride in 50-mmol/L Tris at pH 7.6 and 0.01% hydrogen per-oxide. Sections were counterstained with hematoxylin andphotographed with a Zeiss Ultraphot photoscope. To evalu-ate the specificity of the antibodies, sections were incu-bated with nonimmune mouse serum substituted for theprimary antibodies. Immunostaining for MMP-1, MMP-2,MMP-3, and MMP-9 was considered positive when cyto-plasmic and stromal staining was observed. Immunostain-ing for MT1-MMP and MT2-MMP was considered posi-tive when membrane staining was observed.



ticle, we investigated MMP expression in the altered lim-bal epithelial cells. The data are consistent with these cellscontributing to the pathogenesis of pterygia by secretingMMPs, which promote the invasion by the pterygia andthe dissolution of Bowman’s layer.37

RESULTS

As shown in Figure 1 and Table 1, the specimen of nor-mal conjunctival and limbal tissue (patient CF) did notdisplay immunostaining for any of the MMPs in the epi-thelial basal cells. However, significant cell surface im-munostaining was present with MT1-MMP, and slightstaining for MMP-1 and MMP-9 was seen in the stroma(Figure 1A, D). In contrast to the fresh surgical speci-mens, the 2 cadaver specimens (Table 1) immuno-stained with most MMPs in the epithelial basal cells andstroma. For example, in the 2 specimens of normal ca-daver conjunctiva, limbus, and cornea (patients C34 andC35), immunostaining by the 2 membrane-type MMPs(MT1-MMP and MT2-MMP) was primarily restricted tothe membranes of the basal epithelial cells in the cor-nea, limbus, and conjunctiva and was not present in thestromal compartments of the tissues. Immunostaining ofMMP-9 also was restricted to the membranes of basal epi-thelial cells in the cornea, limbus, and conjunctiva, butin addition, MMP-9 was detected in the stroma of the con-junctiva, limbus, and cornea. Immunostaining for MMP-2was present in the epithelium and stroma of the limbusand cornea but was not detected in the epithelium of theconjunctiva. The stroma of the conjunctiva was variablypositive for MMP-2. Staining for MMP-3 was highly re-stricted to the epithelium and stroma of the cornea andwas not detected in the conjunctiva or limbal tissues. Stain-

ing for MMP-1 was present in the epithelium of the cor-nea and variably present in the corneal stroma and lim-bal epithelium.

All 10 pterygia specimens (9 primary and 1 recur-rent) immunostained with most of the 6 MMPs studied(Table 2). Immunostaining by the MMPs was consis-tently high in the invading limbus epithelium in the al-tered limbal basal cells and in the adjacent corneal andconjunctival epithelia, which were infiltrated by invad-ing altered limbal basal cells (Table 2 and Figures 2,3, and 4). Matrix metalloproteinase 1 was particularlyprominent in the epithelial cells of the invading limbalepithelium (10/10), the adjacent corneal (9/10), and con-junctival epithelial cells (10/10) (Table 2 and Figures 2,3, and 4). The other MMPs were present in the epithe-lium of about 8 of 10 pterygia specimens. In the recur-rent pterygium, we found a single layer of cuboidal cells,which immunostained with all 6 MMPs, spreading on topof the surface of terminally differentiated squamous cells(data not shown). In addition, MMP expression oc-curred in some corneal stromal sections at cut, broken,or crushed areas (Figures 2 and 4). Figure 2G is inter-esting (a higher magnification of Figure 2A) in that itshows staining of MMP-1 in both the basal epithelial cellsand the epithelial side of Bowman’s layer.

Matrix metalloproteinase 1 was found to be the mostfrequently expressed MMP by fibroblasts in pterygia(Table 2 and Figure 4). Matrix metalloproteinase 1 wasoften present in fibroblasts at the dissolved edge of Bow-man’s layer (7/10) and in lobules of pingueculae (6/10)and less frequently present in fibroblasts under the mi-grating limbus (3/10). Fibroblasts found at the dis-solved edges of Bowman’s layer (7/10) and in areas of fi-broblast islands frequently immunostained with MMP-1

Table 1. MMP Expression in Fresh Normal Conjunctiva and Limbus (Patient CF); Cadaver Conjunctiva, Limbus,and Cornea (Patients C34 and C35); and the Area of Elastotic Degeneration (Pinguecula in Patient C35)*

MMP Patient

Conjunctiva Limbus Cornea†Elastotic

Degeneration‡ FibroblastsEpithelium Stroma Epithelium Stroma Epithelium Stroma

MMP-1 CF − − − − −C34 − − (+) − (+) (+) −C35 − − − − +B − + −

MMP-3 CF − − − − −C34 − − − − (+) (+) −C35 − − − − (+) (+) + −

MMP-2 CF − − − (+) −C34 − − (+) + (+) (+) −C35 − + (+) + +B − ++ −

MMP-9 CF − − − − −C34 +B + +B + +B − −C35 +B + +B + +B − ++ −

MT1-MMP CF − − (+)S − −C34 +B − +B − +B − −C35 +B − +B − +B − ++ −

MT2-MMP CF − − − − −C34 +B − (+)B − ++B − −C35 (+) − (+) − + − + −

*MMP indicates matrix metalloproteinase; MT1, membrane type 1; MT2, membrane type 2; −, negative staining; (+), some positive staining areas within specimen;B, basal cell staining only; +, mild staining; ++, moderate staining; and S, squamous epithelial cell staining only.

†Specimen CF had no cornea to stain.‡Only specimen C35 had areas of elastotic degeneration.



(Figure 4). In addition, MMP-1 was expressed by 9 of10 altered limbal basal cells in cornea over Bowman’s layer(Figures 2-4) and frequently stained Bowman’s layer be-neath these basal cells (Table 2).

Areas of elastotic degeneration (pingueculae) usu-ally immunostained for all 6 MMPs (Table 3 and Fig-ure 3), although the fibroblasts in these areas mainly im-munostained for MMP-1 and MMP-3.

A B

C D

E F

Figure 1. Matrix metalloproteinase (MMP) immunostaining in normal limbus epithelium. No MMP staining of basal or suprabasal cells (brown pigmentin some basal cells is melanin). Membrane type 1 (MT1)–MMP staining of some squamous cells. Mild staining of desquamating surface cells (A, C, D, F)and stroma (A, D). Staining of all cut stromal edges: (A) MMP-1, (B) MMP-3, (C) MMP-2, (D) MMP-9, (E) MT1-MMP, and (F) membrane type 2–MMP.Palisades of Vogt are visible in A through D, indicating that the specimens came from the limbal region. Limbal basal cells migrate from left to right(original magnification 3325).

Table 2. Staining of Pterygia With Monoclonal Antibodies to MMPs*

MMP

EpitheliumFibroblasts

Extracellular Matrixin Bowman’s Layer

in PterygiaConjunctivaMigratingLimbus Cornea

UnderMigratingLimbus

At Dissolved Edgeof Bowman’s Layer Islands†

In Fibroblastsin Lobules

of Pingueculae

MMP-1 10/10 10/10 9/10 3/10 7/10 1/1 6/10 9/10MMP-3 8/10 8/10 7/10 0/10 1/10 0/1 2/10 9/10MMP-2 9/10 9/10 10/10 0/10 1/10 0/1 0/10 8/10MMP-9 8/10 8/10 8/10 1/10 0/10 0/1 0/10 8/10MT1-MMP 8/10 8/10 8/10 0/10 0/10 0/1 0/10 4/10MT2-MMP 8/10 8/10 8/10 0/10 1/10 0/1 0/10 3/10

*MMP indicates matrix metalloproteinase; MT1, membrane type 1; and MT2, membrane type 2.†One of the 10 specimens in this study had fibroblast islands.



COMMENT

COLLECTING AND ORIENTING THE SPECIMEN

The identification in pterygia of the migrating limbus withits altered limbal basal cells and their associated acti-

vated fibroblasts depends on the correct surgical collec-tion of the specimen. In addition, proper orientation ofthe specimen is required to demonstrate, with serial crosssections, the key anatomy at the junction where the al-tered limbal basal cells start to invade onto corneal base-ment membrane over dissolving Bowman’s layer.

A B

C D

E

G

F

Figure 2. Corneal invasion by altered limbal basal cells. A group of matrixmetalloproteinase (MMP) immunostaining altered limbal basal cells(pterygium cells, arrowheads) invading the corneal epithelium overBowman’s layer: (A) MMP-1, (B) MMP-3, (C) MMP-2, (D) MMP-9, (E)membrane type 1–MMP, and (F) membrane type 2–MMP. Altered limbalbasal cells invade from left to right (original magnification 3170). Panel G isa higher magnification (3500) of panel A and shows the basal cells stainingnot only for MMP-1 but also on the epithelial side of Bowman’s layer.



NORMAL TISSUE

As in most normal, resting tissues, conjunctival-limbal-corneal epithelial tissues express such small amounts ofMMPs that they are nearly undetectable by techniques suchas immunohistochemistry40-43 (Figure 1). However, MT1-MMP immunostaining was detectable in the surface epi-thelial cells of the fresh normal control specimens (Figure1E). Also, MT1-MMP immunostaining has been found inother normal human tissue.44 In addition, MMP immu-nostaining occurred in some sections at cut, broken, orcrushed areas (Figures 1, 2, and 4), which may be due toan artifactual translocation of the MMPs after surgical

wounding.45,46 From these data, we conclude that carefulcollection of specimens is needed to avoid artifactual MMPstaining due to trauma. In addition, fresh surgical speci-mens are needed, because cadaver eyes, which were 4 to 5hours old, tended to express abnormal levels of MMPs(Table 1).

PTERYGIA

We discovered previously that pterygia consisted of lim-bal epithelial tumor cells that expressed p53 and vimentinand displayed a peculiar development and migration pat-tern.8,9 We also previously demonstrated that the pte-

A B

C D

E F

Figure 3. The 2 tumors of pterygia. (1) The pterygium tumor: matrix metalloproteinase (MMP) immunostaining in altered limbal basal cells (to left of arrowheadsthat point to the dissolved edges of Bowman’s layer), which are invading corneal epithelium over Bowman’s layer (to right of arrowheads). This tumor is locatedabove the space marked with an X (see panel A). (2) The pinguecula tumor: stationary noninvading MMP immunostaining areas of elastotic degeneration(containing altered fibroblasts) that are dragged onto the cornea by the invading pterygium tumor. Staining of the area of elastotic degeneration is seen in panel C;however, no fibroblast staining is observed. The pinguecula is located below the space marked with an X (see panel A). Before tissue processing these 2 tumorswere contiguous. For both tumors, (A) MMP-1, (B) MMP-3, (C) MMP-2, (D) MMP-9, (E) membrane type 1–MMP, and (F) membrane type 2–MMP. Altered limbalbasal cells invade from left to right (original magnification 3100).



rygium cells had characteristics of limbal basal epithelialcells.8,9 In the present study, we found that the altered lim-bal basal epithelial cells of pterygia expressed 6 MMPs ofvarious types similar to other invasive tumors,30-34 and wespeculate that these MMPs are likely to promote cornealinvasion of this tumor and contribute to the dissolution of

Bowman’s layer (Figures 2-4). In addition to migration ofa segment of altered limbal epithelium and local infiltra-tion of pterygium cells within adjacent epithelial tissues,we found in our specimen of recurrent pterygium a pat-tern of surface spread of MMP-expressing cuboidal cellsover terminally differentiated squamous cells, which is simi-

A B

C D

E

G

F

MMP-1 Staining Basal Epithelial Cells

MMP-1 Staining Fibroblasts Dissolved Edges of

Bowman’s Layer

MMP-1 Staining Fibroblast Island

Surgical Cut Edge Staining for MMP-1

Figure 4. Dissolution of Bowman’s layer. Matrix metalloproteinase(MMP) immunostaining in altered limbal basal cells invading cornealepithelium over Bowman’s layer, in activated fibroblasts located at thedissolved edges of Bowman’s layer (small arrowheads), and infibroblast islands (large arrowhead) within dissolved Bowman’s layer:(A) MMP-1, (B) MMP-3, (C) MMP-2, (D) MMP-9, (E) membrane type1–MMP, and (F) membrane type 2–MMP. Altered limbal basal cellsinvade from left to right (original magnification 3250 for A-F). G,Schematic drawing of panel A.



lar to those in one of our previous studies.19 The spread-ing of surface MMP-expressing altered cells has a poten-tial for wider spread than infiltration in the basal layers andcould possibly explain some of the recurrences with auto-grafts or wide excisions and the need for supplemental topi-cal chemotherapeutic eyedrops such as mitomycin.19

MIGRATING LIMBUS EPITHELIUM

The invasion of the cornea by an entire segment of limbalepithelium with altered limbal basal cells can be ex-plained by MMP-2 and MMP-9 expression by these cells.Elevated expressions of both MMP-2 and MMP-9 are knownto dissolve basement membrane components, such ashemidesmosomes, leading to migration and invasion of tu-mor cells.28,29 Consistent with the expression of MMP-2 andMMP-9 by altered limbal cells is elevated MT1-MMP andMT2-MMP expression, since MT1-MMP and MT2-MMPcan activate latent pro–MMP-2 and pro–MMP-9.

DISSOLUTION OF BOWMAN’S LAYER

We previously described 4 different groups of fibro-blasts in pterygia9,19,24: (1) a group of collagen-synthesizing fibroblasts under the migrating limbus nearthe dissolved edge of Bowman’s layer; (2) a group of col-lagenase-synthesizing fibroblasts surrounding the dis-solved edges of Bowman’s layer (Figures 2-4); (3) groupsof collagenase-synthesizing fibroblasts located in is-lands (Ilots de Fuchs)12 (Figure 4) anterior to the lead-ing edges of the pterygium and between corneal base-ment membrane and Bowman’s layer; and (4) groups ofelastotic material–synthesizing fibroblasts in basophilicareas where abnormal elastic-type material was present.

None of these fibroblast groups expressed p53 in pte-rygia, whereas all altered limbal basal cells did synthesizep53,19,23 which suggests that the pterygium cells (ie, al-tered limbal basal epithelial cells) are the main tumor cells.We found that the p53 overexpression colocalized with theMMP expression (data not shown). Most of the fibro-blasts in groups 2, 3, and 4 and a few fibroblasts in group1 expressed mainly MMP-1 and some MMP-3 but almostnone of the other MMPs (Table 2). These findings suggestthat in areas of Bowman’s layer dissolution, fibroblasts aremaking MMPs and most likely play an important role inhelping to dissolve Bowman’s layer. These fibroblasts are

aided in the dissolution of Bowman’s layer by the MMP-1–and MMP-3–expressing limbal basal cells (Figure 4 andFigure 5) as indicated by MMP-1 and MMP-3 immuno-staining of Bowman’s layer in some of the sections. Be-cause the altered limbal basal epithelial cells (the pte-rygium cells) express transforming growth factor b (TGF-b),24,47-49 the adjacent MMP-expressing fibroblasts are mostlikely TGF-b–basic fibroblast growth factor (bFGF) acti-vated cells24 and are not mutationally altered ones19,23,24 (Fig-ure 5).

ALTERED FIBROBLASTS IN ELASTOTICAREA: A SECOND TUMOR

Because fibroblasts in elastotic areas are known to makeabnormal elastic material, they have been considered tobe altered cells.50,51 We found these fibroblasts makingMMP-1 and MMP-3 but none of the other MMPs (Table2). However, since all areas of elastotic degeneration out-side the fibroblasts immunostained for all 6 MMPs (Table3), we assume that the MMPs came from altered fibro-blasts. The altered fibroblast lobules constitute a secondstationary tumor (pingueculae) within the main invad-ing pterygium tumor similar to what is present in otherocular and skin tumors with associated areas in the stromaof elastotic degeneration.52 In all of these UV-inducedgrowths, the main tumor cell type is the epithelial celland not the fibroblast. The fact that tumors consistingof both altered epithelial cells and altered fibroblasts canexist at the same time has been demonstrated in animalexperiments where ocular tissue was treated with long-term, low-dose UV radiation.53,54

Recurrent pterygia that return within a few monthsafter surgery do not usually have sufficient UV expo-sure to develop areas of elastotic degeneration. For thisreason, they were assumed to be different from primarypterygia and to produce an exuberant fibroplasia as a re-sult of an abnormal healing reaction.55 In pterygia recur-ring after several years, we have found elastotic degen-eration in all specimens, including the one reported herein.

THEORY OF PATHOGENESIS OF PTERYGIA

Based on the data presented in this study and our pre-vious reports, we propose a theory for the pathogenesisof pterygia. Albedo UV light56 (Figure 5) causes muta-tions in both the UV-sensitive TP53 tumor suppressorgenes in the parental limbal basal cells and the elastingene of the fibroblasts in the limbal epithelium.19 Be-cause of a damaged p53-dependent programmed cell deathmechanism,20 mutations in other genes are progres-sively acquired. This allows the multistep21 develop-ment of pterygia and limbal tumor cells from p53-expressing limbal epithelial cells. These cells overlie apinguecula of altered fibroblasts that make abnormal elas-totic material and express various MMPs.

Mutations in the TP53 gene or TP53 family in theparental limbal basal cells also result in the overproduc-tion by the pterygium cells of TGF-b via the p53-Rb-TGF-b pathway.24,47 Thus, pterygia are TGF-b–secreting tumors. Excess TGF-b secretion by thepterygium cells can explain many of the tissue changes

Table 3. Staining of Acellular Areas of Pingueculae,Found Within Pterygia, With Monoclonal Antibodiesto MMPs*

MMPStromal Lobules With Wavy

Elastotic MaterialGranular AcellularStromal Lobules

MMP-1 6/7 8/10MMP-3 4/6 8/10MMP-2 5/6 10/10MMP-9 3/6 10/10MT1-MMP 5/6 10/10MT2-MMP 3/6 7/10

*MMP indicates matrix metalloproteinase; MT1, membrane type 1; andMT2, membrane type 2.



and MMP expressions seen in pterygia.24,47-49,57-66 First,pterygium cells (altered limbal basal epithelial cells) pro-duce elevated MMP-2, MMP-9, MT1-MMP, and MT2-MMP, causing dissolution of hemidesmosome attach-ments. Initially, the pterygium cells migrate centrifugallyin all directions onto the adjacent and joined corneal, lim-bal, and conjunctival basement membranes. Because ofthe TGF-b production of these cells, they have a re-duced number of cell layers24,47-49,57 and no tumor massis seen, resulting in an invisible tumor.19 Later, after anentire group of altered limbal basal cells develop and allhemidesmosomes are dissolved under these cells, theymigrate as a suppressed growth onto the cornea fol-lowed by conjunctival epithelium, expressing all 6 MMPsand contributing to the dissolution of Bowman’s layer.In addition, TGF-b synthesized by the pterygium cellscauses increased monocytes and capillaries within the epi-thelial and stromal layers19,24,37,47-49,57-61 (Figure 5). Sec-ond, a group of normal fibroblasts gather under the in-vading limbus epithelium next to the dissolved edges ofBowman’s layer and are activated by a TGF-b–bFGF path-way24 to produce excess MMP-1 and MMP-362 as they helpto dissolve Bowman’s layer. Some of these cytokine-activated fibroblasts migrate anterior to the leading edgesof pterygia between Bowman’s layer and the basementmembrane of the corneal basal cells to form little is-lands of fibroblasts that make MMP-1 and locally helpto dissolve Bowman’s layer24,62 (Figure 4).

The above steps in the formation of a pterygium areseen diagrammatically in Figure 6. Figure 6 shows themigration of the altered limbal basal epithelial cells

(MMP expressing) within the body of the migrating lim-bus and their infiltration into the corneal and conjuncti-val epithelia. Figure 6 also shows the dissolution of Bow-man’s layer under the body of the migrating limbus andthe migration of the adjacent conjunctival epithelial cellsand stromal structures, such as pingueculae, within thepterygium.

CONCLUSIONS

The main body of the tumor that is located in pterygia isfound in the leading edges and is a migrating transpar-ent microscopic piece of altered limbal epithelium. Themigrating limbal epithelium is the occult tumor. If suf-ficient fibroblasts accumulate under the migrating lim-bus at the leading edges, the area can be seen clinicallywith the slitlamp biomicroscope as a gray, glassy cap.9,24

Microscopically, the migrating limbal epithelial tumor isalways located between the dissolved edges of Bow-man’s layer and conjunctival epithelium (as indicated bythe presence of goblet cells). From this migrating lim-bus, altered limbal epithelial basal cells invade centrifu-gally in all directions into adjacent conjunctival, circum-ferential limbal, and corneal epithelia. As the migratingpiece of limbal epithelium moves onto corneal base-ment membrane over Bowman’s layer, the adjacent con-junctival epithelium infiltrated with the altered limbalbasal cells follows, which creates the gross clinical ap-pearance of the pterygium.

Pterygia are tumors of altered limbal basal cells thatsecrete TGF-b and produce various types of MMPs simi-

Limbal Tumor

Pterygium Tumor

Pterygium With Dysplasia

MultistepMutationPathway

Altered Limbal Basal Stem Cells

Altered Limbal Basal Stem Cells

Limbal Epithelial Basal Stem Cells+

Limbal Stromal Fibroblasts

MonocytesMMPsAngiogenesisCapillaries in Limbal Epithelium

Invasion by Altered Limbal Basal Stem Cells

MMP-1MMP-3

Degradation of Fibrillar Collagen I and III in Bowman’s Layer

Denatured Collagen (Gelatin)

Complete Dissolution of Bowman’s Layer

1. Invasion of Bowman’s Layer

2. Fibroplasia3. Elastin

GeneExpression

Activation of Fibroblasts

bFGF

Pinguecula: Fibroblast Tumor1. Elastotic Material2. MMP-1

MMP-33. ? MMP-2

? MMP-9? MT1-MMP? MT2-MMP

Altered Fibroblasts

UV

MMP-2MMP-9

TGF-β

MMP-2MMP-9MT1-MMPMT2-MMP

MMP-1

Figure 5. Possible pathways for development of pterygia. MMPs indicates matrix metalloproteinases; TGF-b, transforming growth factor b; MT1, membrane type1; MT2, membrane type 2; and bFGF, basic fibroblast growth factor. Question marks indicate that not all pinguecula showed elevated MMP-2, MMP-9, MT1-MMP,and MT2-MMP in their fibroblasts (compare Tables 2 and 3).



lar to other invasive tumors. The tumor cell proteases de-grade components of their basement membranes, whichfacilitates invasion. The pterygium cells invading overBowman’s layer produce elevated MMP-1, MMP-2, andMMP-9 expressions, which contribute to the completedissolution of Bowman’s layer, which consists primarilyof collagen fibril types I and III.67 Local fibroblasts areactivated by the TGF-b and bFGF cytokine pathways tohelp complete the dissolution of Bowman’s layer byMMP-1. However, MMP-1 makes only a single cut in in-tact fibrillar collagen (eg, fibrillar collagen types I, II, III,VII, VIII, and X), and then the gelatinases MMP-2 andMMP-9 make successive cuts in the altered type I colla-gen that eventually produces complete destruction of col-lagen strands. As these 2 groups of cells invade into cor-nea, they drag along the adjacent conjunctiva and stromalstructures, such as pingueculae, which consist of focalareas of noninvasive stationary fibroblast tumors syn-thesizing abnormal elastic material and MMPs.

Accepted for publication December 28, 2000.This study was supported in part by grant 61-9783 from

the Kaiser Foundation Research Institute, Oakland, Calif(Dr Dushku), and grant EY05587 from the National Insti-tutes of Health, Bethesda, Md (Dr Schultz).

Drs Dushku and John contributed equally to thisarticle.

We thank Samuel Woo, Illustration Services, Univer-sity of California, Davis, for photography assistance.

Reprints and corresponding author: Nicholas Dushku,MD, Department of Ophthalmology, Kaiser PermanenteMedical Center, 1650 Response Rd, Sacramento, CA 95815(e-mail: [email protected]).

REFERENCES

1. Cameron ME. Geographic distribution of pterygia. Trans Ophthalmol Soc Aust.1962;22:67-81.

2. Taylor HR, West SK, Rosenthal FS, Munoz BM, Newland HS, Emmett EA. Cor-neal changes associated with chronic UV irradiation. Arch Ophthalmol. 1989;107:1481-1484.

3. Cha SB, Shields JA, Shields CL, Wang MX. Squamous cell carcinoma of the con-junctiva. In: Shields JA, ed. International Ophthalmology Clinics. Vol 33, No. 3.Boston, Mass: Little Brown & Co; 1993:19-24.

4. Tabbara KF, Kersten R, Daquk N, Blodi FC. Metastatic squamous cell carcinoma.Ophthalmology. 1988;95:318-321.

5. Clear AS, Chirambo MC, Hutt MSR. Solar keratosis, pterygium, and squamouscell carcinoma of the conjunctiva in Malawi. Br J Ophthalmol. 1979;63:102-109.

6. Spencer WH. Ophthalmic Pathology: An Atlas and Textbook. 3rd ed. Philadel-phia, Pa: WB Saunders Co; 1985:304.

7. Reese AB. Tumors of the Eye. 3rd ed. New York, NY: Harper & Row; 1976:53-55.

ML PterygiumCornea

ML

G

CJ

Sclera

DBL

V

F IF II

F III

Bowman’s Layer

F IV

Bowman’s Layer

MMP B

Corneal Stroma

Original Boundary Between Cornea and Sclera Located at the Original Edge of Bowman’s Layer

Figure 6. Pterygia pathogenesis. Corneal invasion by matrix metalloproteinase (MMP) expressing altered limbal epithelial cells and activation of fibroblasts. CJindicates conjunctiva with goblet cells infiltrated by pterygium cells; DBL, dissolved Bowman’s layer; F I, fibroblasts making abnormal elastotic material (thepinguecula tumor); F II, fibroblasts making collagen and possibly elastic materials; F III, fibroblasts making MMP-1 at dissolved edge of Bowman’s layer; F IV,fibroblasts (fibroblast islands) making MMP-1 at dissolved edges of Bowman’s layer; G, goblet cells; ML, migrating limbus; MMP B, MMP expressing alteredlimbal basal epithelial cells invading cornea and conjunctival epithelium; and V, blood vessels (angiogenesis).



8. Dushku N, Tyler N, Reid TW. Immunohistochemical evidence that pterygia arisefrom altered limbal epithelial basal stem cells [ARVO abstract]. Invest Ophthal-mol Vis Sci. 1993;34:S1013. Abstract 1525.

9. Dushku N, Reid TW. Immunohistochemical evidence that human pterygia origi-nate from an invasion of vimentin-expressing altered limbal epithelial basal cells.Curr Eye Res. 1994;13:473-481.

10. Hogan MJ, Zimmerman LE. Ophthalmic Pathology: An Atlas and Textbook. 2nded. Philadelphia, Pa: WB Saunders Co; 1962:253-254.

11. Duke-Elder S. Diseases of the outer eye. In: Duke-Elder S, ed. System of Oph-thalmology. Vol 8. St Louis, Mo: CV Mosby; 1965:573-582.

12. Cameron M. Pterygium Throughout the World. Springfield, Ill: Charles C Tho-mas Publishers; 1965:125.

13. Kenyon KR, Fogle JA, Grayson M. Dysgeneses, dystrophies and degenerationsof the cornea. In: Tasman W, Jaeger EA, eds. Duane’s Clinical Ophthalmology.Philadelphia, Pa: Lippincott; 1991:1-49.

14. Yanoff M, Fine BS. Ocular Pathology. 2nd ed. Philadelphia, Pa: Harper & Row;1982:332.

15. Brash DE, Rudolph JA, Simon JA, et al. A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci U S A.1991;88:10124-10128.

16. Kress S, Sutter C, Strickland PT, Mukhtar H, Schweizer J, Schwartz M. Carcinogen-specific mutational pattern in the p53 gene in ultraviolet B radiation-induced squa-mous cell carcinomas of mouse skin. Cancer Res. 1992;52:6400-6403.

17. Vogelstein B, Kinzler KW. Carcinogens leave fingerprints. Nature. 1992;355:209-210.

18. Ziegler A, Leffell DJ, Kunala S, et al. Mutation hotspots due to sunlight in thep53 gene of nonmelanoma skin cancers. Proc Natl Acad Sci U S A. 1993;90:4216-4220.

19. Dushku N, Reid TW. p53 Expression in altered limbal basal cells of pingueculae,pterygia and limbal tumors. Curr Eye Res. 1997;16:1179-1192.

20. Kinzler KW, Vogelstein B. Life (and death) in a malignant tumor. Nature. 1996;379:19-20.

21. Weinberg RA. Oncogenes, antioncogenes, and the molecular bases of multistepcarcinogenesis. Cancer Res. 1996;49:3713-3721.

22. Dushku N, Albert DM, Reid TW. The use of PCR to test for human papilloma vi-rus DNA in p53 expressing limbal stem cells of pinguecula, pterygia, and limbaltumors [ARVO abstract]. Invest Ophthalmol Vis Sci. 1998;39:S543. Abstract 2501.

23. Dushku N, Hatcher SLS, Albert DM, Reid TW. p53 Expression and relation toHPV infection in pingueculae, pterygia and limbal tumors. Arch Ophthalmol. 1999;117:1593-1599.

24. Reid TW, Dushku N. Pterygia and limbal epithelial cells: relationship and mo-lecular mechanisms. Prog Retin Eye Res. 1996;15:297-329.

25. Parks WC, Schultz GS. Proteases and protease inhibitors in tissue repair. In:DiZerega G, ed. Peritoneal Surgery. New York, NY: Springer; 2000:101-113.

26. Lee S-B, Li D-Q, Gunja-Smith Z, Liu YQ, Tan DTH, Tseng SCG. Increased ex-pression of MMP-1 and MMP-3 by cultured pterygium head fibroblasts [ARVOabstract]. Invest Ophthalmol Vis Sci. 1999;40:S334. Abstract 1768.

27. Solomon A, Li D-Q, Lee S-B, Teng SCG. Regulation of collagenase, stromelysin,and urokinase-type plasminogen activator in primary pterygium body fibro-blasts by inflammatory cytokines. Invest Ophthalmol Vis Sci. 2000;41:2154-2163.

28. Stetler-Stevenson WG, Aznavoorian S, Liotta LA. Tumor cell interactions withthe extracellular matrix during invasion and metastasis. Ann Rev Cell Biol. 1993;9:541-573.

29. Coussens LM, Werb Z. Matrix metalloproteinases and the development of can-cer. Chem Biol. 1996;8:895-904.

30. Heppner KJ, Matrisian LM, Jensen RA, Rodgers WH. Expression of most matrixmetalloproteinase family members in breast cancer represents a tumor-inducedhost response. Am J Pathol. 1996;149:278-282.

31. Muller D, Breathnach R, Engelmann A, et al. Expression of collagenase-relatedmetalloproteinase genes in human lung or head and neck tumors. Int J Cancer.1991;48:550-556.

32. Shima I, Sasaguri V, Kusukawa J, et al. Production of matrix metalloprotein-ase-2 and metalloproteinase-3 related to malignant behavior of esophageal car-cinoma: a clinicopathologic study. Cancer. 1992;70:2747-2753.

33. Newell KJ, Witty JP, Rogers WH, Matrisian LM. Expression and localization ofmatrix-degrading metalloproteinases during colorectal tumorigenesis. Mol Car-cinog. 1994;10:199-206.

34. Bolon K, Brambilla E, Vandenbunder B, Robert C, Lantuejoul S, Brambilla C.Changes in expression of matrix proteases and of the transcription factor c-Ets-1 during progression of precancerous bronchial lesions. Lab Invest. 1996;75:1-13.

35. Liu YP, Schultz GS, Ren XO, Tan DTH. MMP-2 and MMP-9 levels in pterygia andmatched superior conjunctiva by gelatin zymography [ARVO abstract]. InvestOphthalmol Vis Sci. 1998;39:S756. Abstract 3485.

36. Di Girolamo N, McCluskey P, Lloyd A, Coroneo MT, Wakefield D. Expression ofMMPs and TIMPs in human pterygia and cultured pterygium epithelium cells.Invest Ophthalmol Vis Sci. 2000;41:671-679.

37. Dushku N, John MK, Schultz GS, Reid TW. Pterygia pathogenesis: corneal in-vasion by matrix metalloproteinase (MMP) expressing altered limbal basal stemcells and activation of fibroblasts [ARVO abstract]. Invest Ophthalmol Vis Sci.2000;41:S451. Abstract 2388.

38. Vegh GL, Selcuk TZ, Fulop V, Genest DR, Mok SC, Berkowitz RS. Matrix metal-loproteinases and their inhibitors in gestational trophoblastic diseases and nor-mal placenta. Gynecol Oncol. 1999;75:248-253.

39. Khaw PT, Schultz GS, MacKay SLD, et al. Detection of transforming growth fac-tor-b messenger RNA and protein in human corneal epithelial cells. Invest Oph-thalmol Vis Sci. 1992;33:3302-3306.

40. Parks WC, Sudbeck BD, Doyle GR, Saariahlo-Kere UK. Matrix metalloprotein-ases in tissue repair. In: Parks WC, Mecham RP, eds. Matrix Metalloprotein-ases. San Diego, Calif: Academic Press; 1998:263-297.

41. Di Girolamo N, McCluskey PJ, Lloyd A, Coroneo MT, Wakefield D. Matrix me-talloproteinases and tissue inhibitors of metalloproteinases are expressed in hu-man pterygia and cultured pterygium epithelial cells [ARVO abstract]. Invest Oph-thalmol Vis Sci. 1999;40:771. Abstract 4070.

42. Garrana RMR, Zieske JD, Assouline M, Gipson IK. Matrix metalloproteinases inepithelia from human recurrent corneal erosion. Invest Ophthalmol Vis Sci. 1999;40:1266-1270.

43. Kawashima Y, Saika S, Yamanaka O, Okada Y, Ohkawa K, Ohnishi Y. Immuno-localization of matrix metalloproteinases and tissue inhibitors of metalloprotein-ases in human subconjunctival tissues. Curr Eye Res. 1998;17:445-451.

44. Knauper V, Murphy G. Membrane-type matrix metalloproteinases and cell surface-associated activation cascades for matrix metalloproteinases. In: Parks WC,Mecham RP, eds. Matrix Metalloproteinases. San Diego, Calif: Academic Press;1998:199-218.

45. Hq Y, Azar DT. Expression of gelatinase A and B, and TIMPS 1 and 2 during cor-neal wound healing. Invest Ophthalmol Vis Sci. 1998;39:913-921.

46. Azar DT, Hahn TW, Jain S, Yeh YC, Stetler-Stevensen WG. Matrix metallopro-teinases are expressed during wound healing after excimer laser keratectomy.Cornea. 1996;15:18-24.

47. Dushku N, Reid TW. Immunohistochemical evidence that pterygia originate fromRb and TGFb-expressing, p53 transformed, limbal basal stem cells [ARVO ab-stract]. Invest Ophthalmol Vis Sci. 1995;36:S1027. Abstract 4759.

48. Kria L, Ohira A, Amemiya T. Immunohistochemical localization of basic fibro-blast growth factor, platelet derived growth factor, transforming growth fac-tor-b and tumor necrosis factor-a in the pterygium. Acta Histochem (Jena). 1996;98:195-201.

49. Ren X, Liu YP, Tan DTH, Schultz GS. Elevated expression of TGFb and EGF sys-tem in pterygia tissues and matched superior conjunctiva. Invest Ophthalmol VisSci. 1998;39:5509.

50. Austin P, Jakobiec FA, Iwamoto T. Elastodysplasia and elastodystrophy as the patho-logic bases of ocular pterygia and pinguecula. Ophthalmology. 1983;90:96-109.

51. Chen JK, Tsai RJ, Lin SS. Fibroblasts isolated from human pterygia exhibit trans-formed cell characteristics. In Vitro Cell Dev Biol Anim. 1994;30A:243-248.

52. Spencer WH. Ophthalmic Pathology: An Atlas and Textbook. 3rd ed. Philadel-phia, Pa: WB Saunders Co; 1985:174-176.

53. Blum HF. Carcinogenesis by Ultraviolet Light. Princeton, NJ: Princeton Univer-sity Press; 1959.

54. Buschke W, Friedenwald JS, Moses SG. Effects of ultraviolet irradiation on cor-neal epithelium: mitosis, nuclear fragmentation, post-traumatic cell move-ments, loss of tissue cohesion. J Cell Comp Physiol. 1945;26:147-164.

55. American Academy of Ophthalmology Manual for Basic Clinical Science CourseSection 4: Ophthalmic Pathology and Intraocular Tumors. San Francisco, Calif:American Academy of Ophthalmology; 1999:51-52.

56. Coroneo MT. Pterygium as an early indicator of ultraviolet insolation: a hypoth-esis. Br J Ophthalmol. 1993;77:734-739.

57. Pasquale LR, Dorman-Pease ME, Lutty GA, Quigley HA, Jampel HD. Immuno-localization of TGFb-1, TGFb-2 and TGFb-3 in the anterior segment of the hu-man eye. Invest Ophthalmol Vis Sci. 1993;34:23-30.

58. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM. Trans-forming growth factor type b: rapid induction of fibrosis and angiogenesis invivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci U S A.1986;83:416-417.

59. Liotta LA, Stetler-Stevenson W, Steeg PS. Metastasis suppressor genes. In: De-Vita VT, Hellman S, Rosenberg SA, eds. Oncology. Philadelphia, Pa: Lippincott;1991:85-100.

60. Seiffert P, Sekundo W. Capillaries in the epithelium of pterygium. Br J Ophthal-mol. 1998;82:77-81.

61. Dameron KM, Volpert OV, Tainsky A, Bouck N. Control of angiogenesis in fibro-blasts by p53 regulation of thrombospondin-1. Science. 1994;265:1582-1584.



62. Kay EP, Lee HK, Park KS, Lee SC. Indirect mitogenic effect of transforming growthfactor-b on cell proliferation of subconjunctival fibroblasts. Invest OphthalmolVis Sci. 1998;39:481-486.

63. Van der Zee E, Everts V, Beertsen W. Cytokines modulate routes of collagen break-down. J Clin Periodontol. 1997;24:297-305.

64. Salo T, Lyons JG, Rahemtulla F, Birkedal-Hansen H. Transforming growth fac-tor-b1 up-regulates type IV collagenase expression in cultured human keratino-cytes. J Biol Chem. 1991;266:11436-11441.

65. Fini ME, Girard MT, Matsubara M, Bartlett JD. Unique regulation of the matrixmetalloproteinase, gelatinase B. Invest Ophthalmol Vis Sci. 1995;36:622-633.

66. Giannelli G, Falk-Marzillier J, Schiraldi O, Stetler-Stevenson WG, Quaranta V. In-duction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Sci-ence. 1997;277:225-228.

67. Marshall GE, Konstas AG, Lee WR. Immunogold fine structural localization ofextracellular matrix components in aged human cornea, I: types I-IV collagenand laminin. Graefes Arch Clin Exp Ophthalmol. 1991;229:157-163.

ARCHIVES Web Quiz Winner

Congratulations to the winner of ourJanuary quiz, Hal Kushner, MD, Day-tona Beach, Fla. The correct answerto our January challenge was sub-periosteal hematoma. For a com-plete discussion of this case, see theCase Reports and Small Case Seriessection in the February ARCHIVES

(Sabet SJ, Tarbet KJ, Lemke BN,Smith ME, Albert DM. Subperi-osteal hematoma of the orbit withosteoneogenesis. Arch Ophthalmol.2001;119:301-303).

Be sure to visit the Archives ofOphthalmology World Wide Web site(http://www.archophthalmol.com)and try your hand at our ClinicalChallenge Interactive Quiz. We in-vite visitors to make a diagnosis basedon selected information from a casereport or other feature scheduled to be published in the following month’s printedition of the ARCHIVES. The first visitor to e-mail our Web editors with thecorrect answer will be recognized in the print journal and on our Web site andwill also receive a free copy of the book One Hundred Years of JAMA LandmarkArticles.

Figure 1. Coronal computed tomographic scandemonstrating hematoma in a subperiosteallocation in the right superior orbit.



laboratory sciences pterygia pathogenesis

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