Extracellular Matrix Alterations in Human Corneas With Bullous Keratopathy

Extracellular Matrix Alterations in Human Corneas WithBullous Keratopathy

Alexander V. Ljubimov,* Robert E. Burgeson,^ Ralph J. Butkowski,% John R Couchman,\Rong Rong Wu,§ Yoshifumi Ninomiya,\\ Yoshikazu Sado,\ Ezra Maguen,*Anthony B. Nesburn,* and M. Cristina Kenney*

Purpose. To uncover abnormalities of extracellular matrix (ECM) distribution in human cor-neas with pseudophakic and aphakic bullous keratopathy (PBK/ABK).

Methods. Indirect immunofluorescence with antibodies to 27 ECM components was used onfrozen sections of 14 normal and 20 PBK/ABK corneas.

Results. Fibrillar deposits of an antiadhesive glycoprotein tenascin in the anterior and posteriorstroma, epithelial basement membrane (BM), bullae and subepithelial fibrosis (SEF) areas,and posterior collagenous layer (PCL) were revealed in diseased corneas. Tenascin inmidstroma, which was observed in some cases, correlated with decreased visual acuity. Innormal central corneas, tenascin was never found. Other major ECM abnormalities in PBK/ABK corneas compared to normals included: discontinuous epithelial BM staining for laminin-1 (aipiyl), entactin/nidogen and fibronectin; accumulation of fibronectin and a l - a 2 typeIV collagen on the endothelial face of the Descemet's membrane; and abnormal depositionof stromal ECM (tenascin, fibronectin, decorin, types I, III, V, VI, VIII, XII, XIV collagen)and BM components (type IV collagen, perlecan, bamacan, laminin-1, entactin-nidogen,fibronectin) in SEF areas and in PCL.

Conclusions. This study provides a molecular description of an ongoing fibrosis on the epithe-lial, stromal, and endothelial levels in PBK/ABK corneas. These fibrotic changes may followinitial endothelial damage after cataract surgery, may be caused by the upregulation of fibro-genic cytokines, and may play a significant role in the progression of bullous keratopathy.Invest Ophthalmol Vis Sci. 1996; 37:997-1007.

X seudophakic (PBK) and aphakic (ABK) bullous ker-atopathy are complications of cataract surgery with(PBK) or without (ABK) intraocular lens placement.Together, they are a leading indication for cornealtransplantation.1 Both are characterized by chroniccorneal edema and loss of transparency. Progressivestromal thickening seems to be caused by the intrastro-

From the *Ophthalmology Research Laboratories, Cedars-Sinai Medical Center,UCLA Medical School Affiliate (Los Angeles, CA); the fMGH-Harvard CutaneousBiology Research Center, Massachusetts General Hospital (Charlestown, MA); theXINCSTAR Corporation (Stillwater, MN); the ̂ Department of Cell Biology,University of Alabama at Birmingham; the \\Okayama University Medical Schooland the ̂ Shigei Medical Research Institute (Okayama, Japan).Presented in part at the annual meeting of the Association for Research in Visionand Ophthalmology, Fort Lauderdale, Florida, May 1995.Supported by National Institutes of Health grants EY10836 (MCK), AR36457(JRC), the Skirball Program in Molecular Ophthalmology (AVL), the DiscoveryFund for Eye Research (AVL, MCK), and the Helen Keller Eye Research Foundation(RRW). JRC is an Established Investigator of the American Heart Association.Proprietary interest category: N.Reprint requests: Alexander V. Ljubimov, Cedars-Sinai Medical Center, Davis-5069, 8700 Beverly Boulevard, Los Angeles, CA 90048.

mal accumulation of fluid with formation of fluid-filled lakes.2'3 The epithelial edema causes "blisters"or bullae on the epithelial surface that can rupture,cause pain, and interfere with vision. In advancedcases, subepithelial fibrosis (SEF), formation of a pos-terior collagenous layer (PCL; also known as retrocor-neal fibrous membrane) between the epithelial celllayer and Descemet's membrane (DM), and cornealvascularization can occur. Clinically, pathologically,and histologically, PBK and ABK are similar to eachother4"6 and are categorized separately based only onthe presence or absence of an intraocular lens. Thus,we think it reasonable to refer to them here as onedisease (PBK/ABK).

Chronic edema in PBK/ABK corneas results fromdecreased ability of endothelial cells to remove fluid.This is caused by a persistent decrease in the endothe-lial cell number after the cell damage during cataract

Investigative Ophthalmology & Visual Science, May 1996, Vol. 37, No. 6Copyright © Association for Research in Vision and Ophthalmology 997

998 Investigative Ophthalmology & Visual Science, May 1996, Vol. 37, No. 6

surgery,7"9 to an alteration of the pumping capacityof these cells,7'8'10 or both. The damaged endotheliumcan close the initial defect over time, but, in somecases, the defect persists, leading to the developmentof PBK/ABK. As a potential therapy, transplantationof normal corneal endothelium onto its basementmembrane (DM) has been considered.11'12 However,normal endothelium grows well on normal DM butdoes not spread and grow well on PBK DM.13 Thediseased DM thus provides a poor substrate for thecells. A reduction of endothelial cell number may bean important early event in the PBK/ABK develop-ment. Later, changes of the cell phenotype may occur,leading to the formation of a growth-restricting extra-cellular matrix (ECM) that precludes endothelial rees-tablishment.

Endothelial cell dysfunction also may contributeto corneal edema in PBK/ABK. These cells maintaincorneal hydration through the Na+/K+ ATPase pump.Corneal endothelial cells in PBK/ABK have reducedpump site density compared to normals.10 This func-tional impairment may be a secondary event in PBK/ABK.

Several lines of evidence emphasize the impor-tance of ECM changes in PBK/ABK development:

1. Typically, PBK/ABK corneas develop ECM accu-mulation at the endothelial (PCL) and the epi-thelial (SEF) levels.4"6

2. Corneal epithelial basement membrane (BM) inPBK/ABK lacks fibronectin, laminin, and typeIV collagen,14 which might reduce cell adhesionand facilitate bullae formation.

3. PBK DM shows altered collagen content and lec-tin binding15 and, contrary to normal DM, doesnot support cell growth,13 suggesting structuralalterations, compositional alterations, or both.

Despite these findings, the ECM of PBK/ABK cor-neas has not been studied systematically.

We report here on the detailed characterizationof the ECM and BM composition of PBK/ABK corneaswith antibodies to 27 individual ECM components.Compared to normal corneas, there was an accumula-tion of stromal tenascin and alterations of epithelialBM and DM. We also describe the molecular and cellu-lar composition of PCL and SEF areas. Our resultsprovide the documentation of significant fibroticchanges in PBK/ABK corneas on the molecular level,even in cases in which no PCL or SEF could be found.We think PBK/ABK not only is a disease of chronicedema but has an ongoing fibrosis that plays an im-portant role in its pathology.

MATERIALS AND METHODS

Normal adult human corneas (n = 14) were obtainedwithin 36 hours of death from the National Disease

Research Interchange (Philadelphia, PA). PBK/ABKcorneas (n = 20, Table 1) were obtained within 20hours of penetrating keratoplasty. In each case, thehistologic diagnosis was verified by a pathologist. Cor-neal embedding in OCT compound (Miles, Elkhart,IN), sectioning, indirect immunofiuorescence, andphotography were performed as described.16 Resultsof routine specificity controls16 were negative. Statisti-cal analysis was performed using two-sided Fisher'sexact test.

Antibodies to human al-ab chains of type IVcollagen; to a l (A), «2 (M), aS (K), /ft (Bl), j32 (S),/33 (kalinin Bl), and yl (B2) chains of laminin; toentactin/nidogen, types VII, XII, and XIV collagen,perlecan core protein domain IV (clones A7L6 andC11L1); and to fibronectin have been described indetail.16"18 Affinity-purified polyclonal antibodies torecombinant rat bamacan (basement membranechondroitin sulfate proteoglycan19) core protein,cross-reacted with human, were produced in rabbits(Couchman et al, manuscript in preparation). Amonoclonal antibody to the aS chain of human typeVI collagen (clone 3C4) was a gift from Dr. E. Engvall(Lajolla Cancer Research Foundation, Lajolla, CA).Polyclonal antibodies to human types I, III, and Vcollagen were obtained from Southern Biotechnology(Birmingham, AL); polyclonal antibodies to humandecorin and a monoclonal antibody to human tenas-cin (clone TN-2) were obtained from Chemicon Inter-national (Temecula, CA); a monoclonal antibody tohuman vimentin (clone Vim 3B4) was obtained fromBoehringer Mannheim (Indianapolis, IN); a mono-clonal antibody to cellular fibronectin (clone IST-9)was obtained from Sera-Lab (Crawley Down, UK); amonoclonal antibody to the a l chain of bovine-hu-man type VIII collagen (clone 9H3) was obtained fromSeikagaku America (Rockville, MD); a polyclonal anti-body to human von Willebrand factor, a mixture ofmonoclonal antibodies to human keratins (pankera-tin cocktail), and a monoclonal antibody to human a-smooth muscle actin (clone 1A4) were all obtainedfrom Sigma Chemical (St. Louis, MO). Cross-species-adsorbed fluorescein- and rhodamine-conjugated sec-ondary antibodies were obtained from Chemicon In-ternational.

RESULTS

All PBK/ABK corneal buttons studied representedonly the central corneal part, so their epithelial BMstructure was compared with that of normal centralcorneas only (see16 for differences of the epithelial BMcomposition in normal central cornea and limbus). InPBK/ABK corneas and failed corneal grafts of pseu-dophakic-aphakic eyes (Table 1), similar ECM and

Extracellular Matrix of Bullous Keratopathy Corneas 999

TABLE l. PBK/ABK Patient History

PatientNumber

123456789

1011121314151617181920

Age(years)

7574737482698269865679898083815757796878

Sex

FMMMFMFFFMMFFMFFMFMM

Time Between CataractRemoval and CornealTransplantation(years)

20.7f

12f13f10381

19J35

246t

124

12836

10

Diagnosis

PBKFailed graft, pseudophakicFailed graft, pseudophakicFailed graft, aphakicPBKPBKPBKPBKPBKtABKABKABKFailed graft, aphakicPBKPBKPBKPBKPBKPBKPBK

VisualAcuity*

HMHMHMHMCFCFCFCFCFCFCFCFCF20/150200/40020/5020/6020/400200/10020/150

Tenascin inMidstroma

•+•

+++-t-

—

—

—

-

—

—

—

+——----—

HM = patient recognizes hand movements; CF = patient can count fingers; + = present; — = absent; ± = local and/or weakexpression.* Before last corneal transplantation.f Time between last two consecutive corneal transplantations.% The patient was aphakic for 8 years and then pseudophakic for 11 years.

BM changes were observed; therefore, the data onthese two groups were combined.

Tenascin

Normal central corneal epithelial BM, stroma, andDM were negative for tenascin (Figs. 1A, 1C). In con-trast, all PBK/ABK corneas had fibrillar tenascin de-posits in the anterior stroma beneath Bowman's layer(Fig. IB) and in the posterior stroma adjacent to theDM (Fig. ID). Tenascin also was present in parts ofepithelial BM and the DM (not shown), in areas ofbullae formation (Fig. IB), SEF, and PCL (Fig. ID). Insome cases, usually more advanced (Table 1), tenascindeposits were seen in the midstroma as well. It shouldbe noted that in normal limbus, tenascin was foundin the stroma and only in a part of limbal blood vessels(not shown). This vessel staining pattern is similar tothat observed in other tissues for tenascin-C but differsfrom that of another tenascin variant, tenascin-X, al-ways associated with blood vessels.20 These data sug-gest that our antibody recognized the ubiquitous ten-ascin variant, tenascin-C.

Type IV Collagen Chains

In all PBK/ABK corneas, the staining for al(IV) anda2(IV) chains was observed on the endothelial faceof the DM, often together with the staining of the

stromal face (Fig. 2B). This was in contrast to normalage-matched corneas in which these chains usuallywere found on the stromal face of the DM (Fig. 2A).In more than half of PBK/ABK corneas, the epithelialBM was partially positive for these chains, a patternthat was never seen in normal central corneas (notshown). The distribution of the a$(TV) -a6(IV) chainswas similar to that in normal corneas, with infrequentlocal discontinuities of the epithelial BM (not shown).

Using a new panel of monoclonal antibodies toall six a (IV) chains,18 we have expanded our recentresults16 on the heterogeneity of a (IV) chain distribu-tion in the epithelial BM of central cornea, limbus,and conjunctiva. Both the a5(IV) and the a6(IV)chains were found in the central, limbal, and conjunc-tival [weaker a5(W) staining than in the limbus] re-gions of epithelial BM. Staining for a3(IV) and a4(IV)chains was continuous in the central cornea and dis-continuous or absent in the limbus and proximal con-junctiva,16 but it could reappear in distal conjunctivaon sections denatured in 6 M urea. In the DM, a6(IV)chain was seen on the endothelial face, as werea3(TV)-a5(W) chains. Unlike the latter three chains,a6(IV) chain was not found around keratocytes.

FibronectinThe PBK/ABK corneas had local fibrillar accumula-tions of fibronectin (including cellular isoform) in the


FIGURE l. Tenascin in normal and PBK/ABK corneas, Nostaining is seen in normal corneas in the anterior (A) andposterior (C) parts. In PBK/ABK corneas, tenascin fibrilsare found in the anterior stroma and beneath epithelial cellsin a bulla (B), as well as in the posterior stroma, on theendothelial face of the DM, and in die PCL <D). E = epithe-lium; S = stroma; B = Bowman's layer; DM — Descemet'smembrane; P = posterior collagenous layer, (arrowhead)Space between detaching epithelium and Bowman's layerin a bulla area. Bar = 40 /mi.

anterior stroma (Table 2). The stromal DM face wasnegative, but the endothelial face was positive (Fig.2D), unlike the normals that displayed a reverse stain-ing pattern (Fig. 2G). The PBK/ABK epithelial BMshowed discontinuous staining (not shown); SEF areas(Fig. 4F) and PCL (Fig. 2D) were positive.

Laminin Chains

The PBK/ABK corneas (Table 2) displayed muchweaker than normal and often discontinuous stainingof the epithelial BM for laminin-1 (al/31yl, or A-Bl-B2) chains (Figs. 2E, 2F). In contrast to this, laminin-5 (kalinin-nicein) was preserved in the epithelial BMof most PBK/ABK corneas, as was another anchoringfibril protein, type VII collagen (not shown). Sparsedeposits of laminin-1 were seen in SEF areas (Fig. 4E)and in the PCL (not shown). In the DM, laminin-1had normal distribution. Laminin chains a2 (M) and/?2 (S), normally absent from central corneal epithe-lial BM,16 did not appear in PBK/ABK corneas (notshown).

FIGURE 2. DM and epidielial BM alterations in PBK/ABK cor-neas. (AJB)> type IV collagen (staining with an antibody to theal—«2 triple helical domain); (C,D), fibronectin; (E,F), laminin(31 (Bl) chain in normal (A.C.E) and diseased (B,D,F) corneas.Note die appearance of al—a2 type IV collagen (B) and fibro-nectin (D) on the endodielial face of the DM and in the PCL,as well as a weak, discontinuous staining of the epithelial BMfor laminin /?1 chain (F) in PBK/ABK corneas. Fibronectindisappears from the stromal face of the DM in diseased corneas(compare D,C). E = epithelium; S = stroma; B = Bowman'slayer; P = posterior collagenous layer. Bar = 40 /Am.

Entactin/NidogenEntactin/nidogen staining in PBK/ABK (not shown)was similar to that for laminin-1—that is, it was discon-tinuous and often absent from the epithelial BM andremained normal in the DM (Table 2). SEF areas andPCL (Fig. 5C) contained some entactin/nidogen.


TABLE 2. Major ECM Abnormalities in PBK/ABK Corneas

ECMComponent

Tenascin

al-a2 type IVcollagen

Fibronectin

Laminin-1Entactin/

nidogenBamacanDecorinType VIII

collagenType XIV

collagen

Epithelial BM

Partially positive

Partially positive

Partially negative

Partially negativePartially negative

No change*No change*Occasionally

positiveNo change*

Stromal ECM

Anterior andposteriordepositsf

Anterior deposits

Anterior deposits

No change*No change*

No change*No change*No change* in

most casesNo change*

DM

Positive

Endothelial facepositive

Stromal facemostlynegative

No change*No change*

No change*No change*Endothelial face

positiveNo change*

SubepithelialFibrosis

Positive

Positive

Positive

Mostly negativeMostly negative

Weakly positivePositivePositive

Positive

PCL

Positive

Positive

Positive

Weakly positiveWeakly positive

Weakly positivePositivePositive

Positive

ECM = extracellular matrix; BM = basement membrane; DM = Descemet's membrane; PCL = posterior collagenous layer.* Compared with normal corneasf In some more advanced cases, tenascin deposits in midstroma were also found (see Table 1).

BamacanThis proteoglycan, revealed by the core protein anti-body, was found around keratocytes, endothelial cells(Fig. 3A), and limbal blood vessels in the normal cor-neas. This was unusual because in other tissues, bama-can exclusively associated with BMs.19 In PBK/ABKcorneas, it also was seen on the endothelial face ofthe DM and in the PCL (Fig. 3B) and was associatedwith SEF, including the BM of abnormal epithelium(Fig. 4D).

Decorin

As in rabbit corneas,21 decorin core protein was dis-tributed uniformly in the stroma of normal humancorneas (Fig. 3C) and in Bowman's layer. In diseasedcorneas, it was seen on the endothelial face of theDM, in the PCL (Fig. 3D), and in SEF areas (notshown). The stromal pattern did not change in PBK/ABK (see also3).

Type VIII CollagenIn normal central corneas, it was localized at the stro-mal face of the DM (see also22) and as weakly stainedfine fibrils in the anterior stroma. Limbal stroma andsome of the blood vessels were positive (not shown).In the diseased corneas, type VIII collagen was seenat the endothelial face of the DM, in SEF areas (notshown), and in PCL (Fig. 5A). Stromal distributionusually did not change (not shown).

Type XII CollagenIn normal corneas, type XII collagen was found at thelevel of the epithelial BM, throughout the stroma, on

the endothelial face of the DM (Fig. 3E), and lessintensely in Bowman's layer. In diseased corneas, itwas seen in SEF areas and in PCL (Figs. 3F, 5E) with-out changes in the stroma.

Type XIV Collagen

Only weak and irregular staining around keratocyteswas observed in normal central corneas. In the limbus,stroma, blood vessels, and epithelial BM were positive(not shown). In PBK/ABK corneas, however, this col-lagen was accumulated in SEF areas (not shown) andin PCL (Fig. 5B).

Correlation Between Clinical Features andExtracellular Matrix Changes

Analysis of case histories showed no correlation be-tween the disease duration and severity (evaluated asa degree of vision loss). As shown in Table 1, patientscould be arbitrarily divided into three groups by theirbest-corrected visual acuity before corneal transplanta-tion: recognizing hand movements (severe visionloss), counting fingers, and having visual acuity of 20/400 or better. For most of the ECM changes (includingthe presence and cellularity of SEF and/or PCL),there was no correlation with visual acuity decrease.However, the presence of tenascin in midstroma (Ta-ble 1) was significantly more frequent in group 1 (4of 4) compared to group 2 (2 of 9; P = 0.021) andespecially to group 3 (0 of 7; P = 0.003). There wasalso a tendency, though not significant, to an in-creased incidence of tenascin in midstroma in group2 compared to group 3. The presence of tenascin inmidstroma correlated with visual acuity decrease and


FIGURE3. Bamacan (A,B). decorin (C,D), and type XII colla-gen (E,F) in normal (A,C,E) and diseased (B,D,F) corneas.Note abnormal deposition of bamacan (B), decorin (D),and type XII collagen (F) on the endothelial face of theDM and in the PCL in PBK/ABK corneas. E = epithelium;S = stroma; P = posterior collagenous layer. Bar = 40 jj,m.

may reflect a more advanced fibrosis. Midstromal ten-ascin was also a common finding in failed grafts (4 of4), compared to primary PBK/ABK (2 of 16; P =0.003; Table 1).

Extracellular Matrix Composition ofSubepithelial Fibrosis Areas and Bullae inPBK/ABK Corneas

Bullae are regions of fluid accumulation in PBK/ABKcorneas either beneath the epithelium or between theepithelial cells. After the bullae resorb, ECM may bedeposited in these areas. A subepithelial fibrocellularmaterial (pannus) disrupting the epithelial BM and

Bowman's layer was noted previously in PBK/ABK cor-neas.'1'715 In half the PBK/ABK corneas studied here,abnormal ECM deposits with or without cells werefound between the epithelium and the Bowman'slayer. Two types of these subepithelial deposits weredistinguished here. The first one may be termed ECM-type deposit because only ECM without cells was pres-ent between the epithelium and the Bowman's layer(Fig. 4A). This type of SEF had abnormal accumula-tions of fibronectin (including cellular), tenascin, per-lecan, decorin, and types I, III, IV (mostly al-a2chains), V, VI, VIII, XII, and XTV collagen, with somelaminin-1, laminin-5, entactin/nidogen, and bamacan(Figs. 4B to 4E). Staining for stromal components wasmore pronounced than for most BM components (seeFigs. 4B to 4E). The second type of SEF (fibrocellulartype) contained cells embedded in the ECM, whichwas qualitatively the same as in the first type (see Fig.4F). The cells contained vimentin (Fig. 4G) and a-smooth muscle actin (Fig. 4H), typical for myofi-broblasts, but did not stain for keratins (not shownhere). These cells may be the actual source of theabnormal subepithelial ECM. In areas of bullae with-out SEF, the basal surface of detaching epitheliumalso stained for abnormal ECM (Fig. IB). Some ECMaccumulated between epithelial cells or layers (Figs.IB, 4D, 4F) in contrast to the normal corneas.

Posterior Collagenous Layer Composition ofPBK/ABK CorneasThe PCL often is found in PBK/ABK corneas as anadditional fibrous layer between die remnant DM andthe endothelial cells. In our series of PBK/ABK, thePCL was observed in 80% of cases, in accordance withprevious studies.615 PCL contained types VI, XII, andXIV collagen, tenascin, fibronectin, decorin, bama-can, perlecan, some laminin-1 and entactin-nidogen(Figs. ID, 2D, 3B, 3D, 3F, 5A to 5E), in addition tothe previously reported types I, III, IV, V, and VIIIcollagen.1523'24 The presence of tenascin, type XTV col-lagen, bamacan, and decorin may be significant be-cause they were not seen in normal DM but appearedin diseased DM and in the PCL. PCL also containedal-a2 type IV collagen (together with some aS~a6chains) and fibronectin, including cellular isoform(Fig. 2D), that were not observed normally on theendothelial face of the DM.16 Thus, PCL was an accu-mulation of corneal stromal and BM components.This ECM-type of PCL resembled the ECM-type ofSEF. In two cases, well-developed PCLs with myofi-broblasts inside them (Figs. 5D to 5F) were found (seealso25), apparently corresponding to the fibrocellular-type SEF.

DISCUSSIONPBK/ABK remains a leading indication for cornealtransplantation, but there is litde basic information


FIGURE 4. Subepithelial fibrosis in PBK/ABK corneas. (A) Hematoxylin staining of an areawith extracellular matrix-type SEF, No abnormal cells are seen between the epithelium andthe Bowman's layer, (B to E) Type XII collagen (B, same section as in A), type V collagen(C), bamacan (D), and laminin y\ (B2) chain (E) in an extracellular matrix-type SEF. Noteheavy deposits of stromal collagens type V and XII compared to scarce deposits of BMcomponents (bamacan and laminin-1). (F to H) Fibrocellular-type SEF with cellular fibro-nectin deposits (F) and myofibroblasts containing vimentin (G) and a-smooth muscle actin(H). E = epithelium; S = stroma; B = Bowman's layer. Bar = 40 fxm.

concerning this disorder. Studies26 dispute the theorythat intraocular lenses affect the protein levels or com-plement factor C3 or C3a in aqueous humor that couldplay a role in inflammation. The stroma of the PBK/ABK corneas has decreased keratan sulfate content,3'27

but the distribution of types V and VI collagen doesnot change.28 One study described lack of laminin,fibronectin, and type IV collagen in the epithelial BMof PBK/ABK corneas.14 PBK/ABK corneas often havea thickened DM with an accumulation of new fibriUarECM with or without cells (PCL) between the endo-thelium and the DM.23'25'29 This ECM contains types Iand V collagen,24 as well as some type III collagen,23

that are not present in this location in normal corneas.Here, the evidence for ongoing fibrosis in PBK/

ABK corneas is provided. In 16 of 20 cases, a fibroticPCL containing stromal ECM and BM componentswas seen, and in 10 of 20 cases, SEF with ECM deposi-tion was observed. It should be noted that the abnor-

mal fibrotic deposition of ECM proteins was detectedeven in PBK/ABK corneas without evidence of SEF orPCL formation. The most conspicuous alteration ofECM pattern in PBK/ABK corneas was an inductionof stromal tenascin.

Tenascin is a large hexameric glycoprotein com-posed of multiple domains.30"33 The tenascin familycomprises three proteins that are different gene prod-ucts3233: tenascin-C (the first discovered), tenascin-R(restrictin), and tenascin-X (X gene product). Theymay play a role in cell migration and proliferation,tissue repair, and oncogenesis.30'31'33 In addition, alter-native splicing of fibronectin type Ill-like (FN-HI) re-peats generates a number of isoforms.34 Tenascin isantiadhesive. Cells may attach to it but do not spreadwell.35 The cell-binding sites are in the FN-III domainsII-VI and in the fibrinogen-like domain, whereas theepidermal growth factor-like domain is antiadhe-sive.35"37 Tenascin can weaken cell-substrate attach-

1004 Investigative Ophthalmology 8c Visual Science, May 1996, Vol. 37, No. 6

FIGURE 5. Posterior collagenous layer in PBK/ABK corneas.(A to C) Extracellular matrix-type PCL containing types VIII(A) and XIV (B) collagen and entactin/nidogen (C). (D toF) Fibrocellular-type PCL containing types VI (D) and XIIcollagen (E) and numerous myofibroblasts positive for a-smooth muscle actin (F). S = stroma; P = posterior collage-nous layer. Bar = 40 jxm.

ment and promote cell detachment by loss of intercel-lular contacts and downregulation of focal adhe-sions.303338'39 Its expression can be induced byfibrogenic cytokines,40"42 such as transforming growthfactor-/3l, basic fibroblast growdi factor, and platelet-derived growth factor-BB.

In normal cornea, tenascin is present only in thelimbal stroma and BM.43M In corneal wound healing,

it appears near the wound edges after fibronectin anddisappears more slowly than fibronectin.45"47 Its cellu-lar origin in the wounded corneas is unknown. In allPBK/ABK corneas studied here, abnormal tenascindeposits were found in anterior and posterior stroma,in the epithelial BM-Bowman's layer, and in the DM,as well as in SEF areas and in PCL. In more advancedcases, tenascin also appeared in midstroma, which cor-related with loss of visual acuity (Table 1). In all butone of the studied corneas, the time between cataractsurgery and corneal transplantation for subsequentPBK/ABK varied from 1 to 24 years (Table 1). Inrabbit corneal wound healing, tenascin can be de-tected in the scar area for only as long as 1 year afterinjury.45"47 These data suggest that tenascin accumula-tion in PBK/ABK corneas probably is not part of cor-neal wound healing associated with cataract surgeryand is more likely triggered later in the course of thedisease.

Tenascin may be upregulated in PBK/ABK by thefollowing mechanism. In the diseased corneas, stromalfluid accumulation and a resultant swelling may createa significant mechanical stress to stromal keratocytes.In this situation, the cells could attempt to relievethe strain and preserve normal stromal structure bycontracting their surrounding collagen. In culturedfibroblasts, such collagen contraction has been shownspecifically to induce production and deposition oftenascin.48 It is thus possible that tenascin induction inPBK/ABK corneas could be triggered by mechanicalstress. Some data seem to support this assumption.The degree of edema in PBK/ABK-failed cornealgrafts is higher dian in primary PBK/ABK49 Ac-cording to the stress hypothesis, the failed graftsshould have more stromal tenascin tfian primaryPBK/ABK corneas. Indeed, all four failed grafts stud-ied here showed marked staining for tenascin inmidstroma, in addition to anterior and posteriorstroma. However, of 16 corneas widi primary PBK/ABK, staining for tenascin in midstroma was notedonly in two, and it was weaker than in failed grafts(Table 1).

Another salient feature of PBK/ABK corneas wasan alteration of BM structure. In accordance with pre-vious data,14 there was a reduction (up to a completeabsence) of fibronectin, laminin-1, and entactin-ni-dogen in the epithelial BM. This could lead to aweaker epithelial adhesion to the Bowman's layer andcould facilitate bullae formation. The DM alterationsin PBK/ABK corneas were also found. They primarilyconcerned a l - a 2 type IV collagen and fibronectin.In normal-aged corneas, both proteins usually wereseen on the stromal face of the DM.16 However, inPBK/ABK corneas, they were found on the endothe-lial face of the DM, irrespective of the presence orabsence of a PCL (Figs. 2B, 2D; Table 2)/It is unclear


which cells are responsible for these changes in PBK/ABK. They could be keratocytes, which are presumablythe normal source of fibronectin and of al-a2 typeIV collagen in the DM. On the other hand, endothelialcells, which apparently do not deposit these proteinsin normal corneas,16 could acquire an altered ECMpattern during PBK/ABK development. It is notewor-thy that cultured corneal endothelium responded tothe endogenous basic fibroblast growth factor by mod-ulating ECM production from type IV to types I andV collagen.50'51 Because basic fibroblast growth factorcan induce fibrosis, it might contribute to the changesof the DM composition in PBK/ABK.

A common feature of PBK/ABK corneas is theformation of PCL and of bullae with subsequent SEF.This article provides a detailed description of the ECMmolecular composition in these areas. In both cases,abnormal ECM had a similar composition and was amixture of stromal (tenascin, decorin, and collagentypes I, III, V, VI, VIII, XII, and XIV) and BM compo-nents (type IV collagen, laminin-1, entactin-nidogen,perlecan, bamacan, and fibronectin). Importantly,PCL contained tenascin in all cases. This antiadhesiveglycoprotein could provide an ECM environment forthe endothelial cells in which they would be unableto attach normally, to spread, and to maintain normalcorneal hydration (see also13). Studies are under wayto determine which cells produce tenascin in thePBK/ABK corneas, which cytokines trigger its induc-tion, and which tenascin splice variants are expressed.

Staining for stromal components was considerablystronger in PCL and in SEF than for most BM compo-nents (see, for example, Fig. 4), suggesting that cellsthat normally produce stromal ECM (apparently kera-tocytes) may contribute to its abnormal accumulationin PBK/ABK corneas. However, in the stroma of suchcorneas, the major ECM components did not seem tobe affected. Possibly, the epithelial and endothelialcell phenotype in PBK/ABK could be modulated to-ward the production of an ECM (in ECM-type SEFand PCL) normally present in the stroma (seealso50'51), with decreased deposition of BM compo-nents. This could be an attempt of the cells to increasetheir adhesion to the ECM in edematous conditionsand preserve the corneal integrity. In fibrocellular-type SEF and PCL, myofibroblasts may be the sourceof abnormal ECM. In SEF, myofibroblasts seem tooriginate from stromal elements but not from epithe-lial cells or their limbal precursors because they do notcontain epithelial markers (keratins). In fibrocellular-type PCL, the source of its fibroblast-like cells (myofi-broblasts) may be modulated endothelium.52

Following the primary defect in PBK/ABK cor-neas, possibly at the endothelial level, an active fibrosisensues that definitely contributes to the disease pro-gression. In fact, there is a correlation between the

decrease in visual acuity of patients and the appear-ance of tenascin in midstroma that may be a markerof advanced fibrosis. The major ECM abnormalitiesfound in PBK/ABK corneas cannot be explained eas-ily by an altered fluid balance. Vision-threateningchanges may result from abnormal production anddeposition of ECM by dysfunctional cells, which mayexacerbate the disease. By inference from other sys-tems, excessive ECM production in PBK/ABK corneascould be modulated by fibrogenic cytokines, trans-forming growth factor-/?l(— /32), basic fibroblastgrowth factor, or platelet-derived growth factor. Direc-tional modulation of the levels of involved cytokinesmay make it possible to normalize corneal ECM andto slow down or stop the disease progression, as dem-onstrated for fibrotic diseases in other organs.53 Onlythen, transplantation of normal endothelium to re-store its function could become feasible.

Key Words

basement membrane, bullous keratopathy, corneal edema,extracellular matrix, immunocytochemistry

Acknowledgments

The authors thank Drs. T. Perl (Corneal Associates of NewJersey, West Orange, NJ) for providing tissue, E. Engvall(La Jolla Cancer Research Foundation, La Jolla, CA) forantilaminin antibodies, A. F. Michael (University of Minne-sota, Minneapolis, MN) for anti-a5(IV) antibody, D. J.Brown (Cedars-Sinai Medical Center, Los Angeles, CA) forvaluable comments, and Z. Huang for expert assistance. An-tibodies to laminin (52 chain, produced by Dr. J. Sanes, andto type IV collagen al-a2 chains, produced by Dr. H.Furthmayr, were obtained from the Developmental StudiesHybridoma Bank, Department of Biology, University of Iowa(Iowa City, IA), under contract NO1-HD-2-3144 from theNICHD.

References

1. Mamalis N, Anderson CW, Kreisler KR, LunderganMK, Olson RJ. Changing trends in the indications forpenetrating keratoplasty. Arch Ophthalmol 1992; 110:1409-1411.

2. Kanai A, Kaufman HE. Electron microscopic studiesof swollen corneal stroma. Ann Ophthalmol 1973; 5:178-190.

3. Quantock AJ, Meek KM, Brittain P, Ridgway AEA,Thonar EJMA. Alteration of the stromal architectureand depletion of keratan sulphate proteoglycans inoedematous human corneas: Histological, imraunochemical and X-ray diffraction evidence. Tissue Cell.1991;23:593-606.

4. Eagle RC, Laibson PR, Arentsen JJ. Epithelial abnor-malities in chronic corneal edema: A histopathologi-cal study. Trans Am Ophthalmol Soc. 1990; 87:107-124.

5. Johnson DH, Bourne WM, Campbell RJ. The ultra-structure of Descemet's membrane: II: Aphakic bul-


lous keratopathy. Arch Ophthalmol. 1982; 100:1948-1951.

6. Grungreiff J, Scholte HW, Kemnitz P. Etiology andmorphology of retrocorneal membranes following mi-crosurgical operations of the anterior eye segment.Klin Monatsbl Augenheilkd. 1988; 193:138-141.

7. Liu GJ, Okisaka S, Mizukawa A, Momose A. Histopath-ological study of pseudophakic bullous keratopathydeveloping after anterior chamber of iris-supportedintraocular lens implantation. Jpn J Ophthalmol. 1993;37:414-425.

8. Numa A, Nakamura J, Takashima M, Kani K. Long-term corneal endothelial changes after intraocularlens implantation. Anterior vs posterior chamberlenses. JpnJ Ophthalmol 1993; 37:78-87.

9. Bourne WM, Nelson LR, Hodge DO. Continued endo-thelial cell loss ten years after lens implantation. Oph-thalmology. 1994; 101:1014-1023.

10. McCartney MD, Robertson DP, Wood TO, McLaug-hlin BJ. ATPase pump site density in human dysfunc-tional corneal endothelium. Invest Ophthalmol Vis Sci.1987;28:1955-1962.

11. Insler MS, Lopez JG. Transplantation of cultured hu-man neonatal corneal endothelium. Curr Eye Res.1986;5:967-972.

12. McCulley JP, Maurice DM, Schwartz BD. Cornealendothelial transplantation. Ophthalmology. 1980; 87:194-201.

13. Raphael B, Lange T, Wood TO, McLaughlin BJ.Growth of human corneal endothelium on alteredDescemet's membrane. Cornea. 1992; 11:242-249.

14. Hsu JKW, Rubinfeld RS, Barry P, Jester JV. Anteriorstromal puncture: Immunohistochemical studies inhuman corneas. Arch Ophthalmol. 1993; 111:1057-1063.

15. Kenney MC, Chwa M. Abnormal extracellular matrixin corneas with pseudophakic bullous keratopathy.Cornea. 1990;9:115-121.

16. Ljubimov AV, Burgeson RE, Butkowski RJ, MichaelAF, Sun TT, Kenney MC. Human corneal basementmembrane heterogeneity: Topographical differencesin the expression of type IV collagen and lamininisoforms. Lab Invest. 1995; 72:461-473.

17. Lunstrum GP, Morris NP, McDonough AM, KeeneDR, Burgeson RE. Identification and partial character-ization of two type Xll-like collagen molecules. J CellBiol. 1991; 113:963-969.

18. Ninomiya Y, Kagawa M, Iyama K, et al. Differentialexpression of two basement membrane collagengenes, COL4A6 and COL4A5, demonstrated by im-munofluorescence using peptide-specific monoclonalantibodies. / Cell Biol. 1995; 130:1219-1229.

19. McCarthy KJ, Abrahamson DR, Bynum KR, St. JohnPL, Couchman JR. Basement membrane-specificchondroitin sulfate proteoglycan is abnormally associ-ated with the glomerular capillary basement mem-brane of diabetic rats. / Histochem Cytochem. 1994;42:473-484.

20. Matsumoto K, Saga Y, Ikemura T, Sakahara T, Chi-quet-Ehrismann R. The distribution of tenascin-X is

distinct and often reciprocal to that of tenascin-C. /Cell Biol. 1994;125:483-493.

21. Zhan Q, Burrows R, Cintron C. Cloning and in situhybridization of rabbit decorin in corneal tissues. In-vest Ophthalmol Vis Sci. 1995; 36:206-215.

22. Tamura Y, Konomi H, Sawada H, Takashima S, Naka-jima A. Tissue distribution of type VIII collagen inhuman adult and fetal eyes. Invest Ophthalmol Vis Sci.1991; 32:2636-2644.

23. Kay EP, Cheung C-C, Jester JV, Nimni ME, Smith RE.Type I collagen and fibronectin synthesis by retrocor-neal fibrous membrane. Invest Ophthalmol Vis Sci.1982:22:200-212.

24. Tuberville A, Wood TO, Hasty K. The 'posterior col-lagenous layer' does contain collagen. ARVO Ab-stracts. Invest Ophthalmol Vis Sci. 1989; 30:336.

25. Waring GO III. Posterior collagenous layer of the cor-nea: Ultrastructural classification of abnormal collage-nous tissue posterior to Descemet's membrane in 30cases. Arch Ophthalmol. 1982; 100:122-134.

26. Tuberville AW, Wood TO. Aqueous humor proteinand complement in pseudophakic eyes. Cornea.1990;9:249-253.

27. Quantock AJ, Meek KM. Proteoglycan distribution inthe corneas of individuals with bullous keratopathy.Biochem Soc Trans. 1990; 18:958.

28. Lee C-F, Yue BYJT, Robin J, Sawaguchi S, Sugar J.Immunohistochemical studies of Peters' anomaly.Ophthalmology. 1989; 96:958-964.

29. Michels RG, Kenyon KR, Maumenee AE. Retrocornealfibrous membrane. Invest Ophthalmol Vis Sci. 1972; 11:822-831.

30. Chiquet-Ehrismann R. What distinguishes tenascinfrom fibronectin? FASEBJ. 1990; 4:2598-2604.

31. Sage EH, Bornstein P. Extracellular proteins thatmodulate cell-matrix interactions: SPARC, tenascin,and thrombospondin. / Biol Cell. 1991; 266:14831-14834.

32. Erickson HP. Tenascin-C, tenascin-R and tenascin-X:A family of talented proteins in search of functions.Curr Opin Cell Biol. 1993; 5:869-876.

33. Lightner VA. Tenascin: Does it play a role in epider-mal morphogenesis and homeostasis? J Invest Dermatol.1994;102:273-277.

34. Kaplony A, Zimmermann DR, Fischer RW, et al. Ten-ascin Mr 220,000 isoform expression correlates withcorneal cell migration. Development. 1991; 112:605-614.

35. Spring J, Beck K, Chiquet-Ehrismann R. Two con-trary functions of tenascin: Dissection of the activesites by recombinant tenascin fragments. Cell.1989;59:325-334.

36. Prieto AL, Andersson-Fisone C, Crossin KL. Charac-terization of multiple adhesive and counteradhesivedomains in the extracellular matrix protein cytotactin./ Cell Biol. 1992; 119:663-678.

37. Joshi P, Chung CY, Aukhil I, Erickson HP. Endothelialcells adhere to the RGD domain and the fibrinogen-like terminal knob of tenascin. / Cell Sci. 1993; 106(part l):389-400.

38. Woods A, Couchman JR. Focal adhesions and cell-


matrix interactions. Collagen Relat Res. 1988;8:155-182.

39. Murphy-Ullrich JE, Lightner VA, Aukhil I, Yan YZ,Erickson HP, Hook M. Focal adhesion integrity isdownregulated by the alternatively spliced domain ofhuman tenascin. / Cell Biol. 1991;115:1127-1136.

40. Tucker RP, Hammarback JA, Jenrath DA, Mackie EJ,Xu Y. Tenascin expression in the mouse: In situ local-ization and induction in vitro by bFGF. / Cell Sci.1993;104(part l):69-76.

41. Sakai T, Kawakatsu H, Ohta M, Saito M. Tenascininduction in tenascin nonproducing carcinoma celllines in vivo and by TGF-beta 1 in vitro. J Cell Physiol.1994;159:561-572.

42. LaFleur DW, Fagin JA, Forrester JS, Rubin SA, SharifiBG. Cloning and characterization of alternativelyspliced isoforms of rat tenascin. J Biol Chem. 1994; 269:20757-20763.

43. Tervo T, van Setten G-B, Lehto I, Tervo K, TarkkanenA, Virtanen I. Immunohistochemical demonstrationof tenascin in the normal human limbus with specialreference to trabeculectomy. Ophthalmic Res. 1990;22:128-133.

44. Tuori A, Virtanen I, Aine E, Salminen L, Uusitalo HM.Expression of tenascin in keratoconus and normal hu-man cornea. ARVO Abstracts. Invest Ophthalmol Vis Sci.1994;35:1358.

45. Latvala T, Tervo K, Mustonen R, Tervo T. Expressionof cellular fibronectin and tenascin in the rabbit cor-

nea after excimer laser photorefractive keratectomy:A 12-month study. BrJ Ophthalmol. 1995;79:65-69.

46. Tervo K, van Setten G-B, Beurman RW, Virtanen I,Tarkkanen A, Tervo T. Expression of tenascin andcellular fibronectin in the rabbit cornea after anteriorkeratectomy. Invest Ophthalmol Vis Sci. 1991; 32:2912-2918.

47. van Setten G-B, Koch JW, Tervo K, et al. Expressionof tenascin and fibronectin in the rabbit cornea afterexcimer laser surgery. Graefe's Arch Clin Exp Ophthal-mol. 1992;230:178-183.

48. Chiquet-Ehrismann R, Tannheimer M, Koch M, etal. Tenascin-C expression by fibroblasts is elevated instressed collagen gels. J Cell Biol. 1994; 127:2093-2101.

49. Lass JH, Greiner JV, Reinhart WJ, Medcalf SK, GlonekT. Phosphatic metabolism and corneal edema. Cornea.1991; 10:346-353.

50. Kay EP, Gu X, Ninomiya Y, Smith RE. Corneal endo-thelial modulation: A factor released by leukocytesinduces basic fibroblast growth factor that modulatescell shape and collagen. Invest Ophthalmol Vis Sci.1993; 34:663-672.

51. Kay EP. Expression of types I and IV collagen genesin normal and in modulated corneal endothelial cells.Invest Ophthalmol Vis Sci. 1989; 30:260-268.

52. Silbert AM, Baum JL. Origin of the retrocorneal mem-brane in the rabbit. Arch Ophthalmol. 1979;97:1141-1143.

53. Border WA, Noble NA. Transforming growth factorP in tissue fibrosis. NEnglJMed. 1994; 331:1286-1292.

Extracellular Matrix Alterations in Human Corneas With Bullous Keratopathy

Documents

Transcript of Extracellular Matrix Alterations in Human Corneas With Bullous Keratopathy