Extracellular Matrix Alterations in Human Corneas with Bullous Keratopathy

Extracellular Matrix Alterations in Human Corneas With Bullous 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 corneas with pseudophakic and aphakic bullous keratopathy (PBK/ABK). Methods. Indirect immunofluorescence with antibodies to 27 ECM components was used on frozen sections of 14 normal and 20 PBK/ABK corneas. Results. Fibrillar deposits of an antiadhesive glycoprotein tenascin in the anterior and posterior stroma, epithelial basement membrane (BM), bullae and subepithelial fibrosis (SEF) areas, and posterior collagenous layer (PCL) were revealed in diseased corneas. Tenascin in midstroma, which was observed in some cases, correlated with decreased visual acuity. In normal 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 al-a2 type IV collagen on the endothelial face of the Descemet's membrane; and abnormal deposition of 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 epithelial, stromal, and endothelial levels in PBK/ABK corneas. These fibrotic changes may follow initial endothelial damage after cataract surgery, may be caused by the upregulation of fibrogenic 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- mal accumulation of fluid with formation of fluid- atopathy are complications of cataract surgery with filled lakes.2'3 The epithelial edema causes "blisters" (PBK) or without (ABK) intraocular lens placement. or bullae on the epithelial surface that can rupture, Together, they are a leading indication for corneal cause pain, and interfere with vision. In advanced transplantation.1 Both are characterized by chronic cases, subepithelial fibrosis (SEF), formation of a pos- corneal edema and loss of transparency. Progressive terior collagenous layer (PCL; also known as retrocor- stromal thickening seems to be caused by the intrastro- neal fibrous membrane) between the epithelial cell layer and Descemet's membrane (DM), and corneal From the *Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, vascularization can occur. Clinically, pathologically, UCLA Medical School Affiliate (Los Angeles, CA); the fMGH-Harvard Cutaneous Biology Research Center, Massachusetts General Hospital (Charlestown, MA); the and histologically, PBK and ABK are similar to each 4 6 XINCSTAR Corporation (Stillwater, MN); the ^Department of Cell Biology, other " and are categorized separately based only on University of Alabama at Birmingham; the \\Okayama University Medical School and the ^Shigei Medical Research Institute (Okayama, Japan). the presence or absence of an intraocular lens. Thus, Presented in part at the annual meeting of the Association for Research in Vision we think it reasonable to refer to them here as one and Ophthalmology, Fort Lauderdale, Florida, May 1995. Supported by National Institutes of Health grants EY10836 (MCK), AR36457 disease (PBK/ABK). (JRC), the Skirball Program in Molecular Ophthalmology (AVL), the Discovery Chronic edema in PBK/ABK corneas results from Fund for Eye Research (AVL, MCK), and the Helen Keller Eye Research Foundation (RRW). JRC is an Established Investigator of the American Heart Association. decreased ability of endothelial cells to remove fluid. Proprietary interest category: N. Reprint requests: Alexander V. Ljubimov, Cedars-Sinai Medical Center, Davis- This is caused by a persistent decrease in the endothe- 5069, 8700 Beverly Boulevard, Los Angeles, CA 90048. lial cell number after the cell damage during cataract

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surgery,7"9 to an alteration of the pumping capacity Research Interchange (Philadelphia, PA). PBK/ABK of these cells,7'8'10 or both. The damaged endothelium corneas (n = 20, Table 1) were obtained within 20 can close the initial defect over time, but, in some hours of penetrating keratoplasty. In each case, the cases, the defect persists, leading to the development histologic diagnosis was verified by a pathologist. Cor- of PBK/ABK. As a potential therapy, transplantation neal embedding in OCT compound (Miles, Elkhart, of normal corneal endothelium onto its basement IN), sectioning, indirect immunofiuorescence, and membrane (DM) has been considered.11'12 However, photography were performed as described.16 Results normal endothelium grows well on normal DM but of routine specificity controls16 were negative. Statisti- does not spread and grow well on PBK DM.13 The cal analysis was performed using two-sided Fisher's diseased DM thus provides a poor substrate for the exact test. cells. A reduction of endothelial cell number may be Antibodies to human al-ab chains of type IV an important early event in the PBK/ABK develop- collagen; to al (A), «2 (M), aS (K), /ft (Bl), j32 (S), ment. Later, changes of the cell phenotype may occur, /33 (kalinin Bl), and yl (B2) chains of laminin; to leading to the formation of a growth-restricting extra- entactin/nidogen, types VII, XII, and XIV collagen, cellular matrix (ECM) that precludes endothelial rees- perlecan core protein domain IV (clones A7L6 and tablishment. C11L1); and to fibronectin have been described in 16 18 Endothelial cell dysfunction also may contribute detail. " Affinity-purified polyclonal antibodies to to corneal edema in PBK/ABK. These cells maintain recombinant rat bamacan (basement membrane 19 corneal hydration through the Na+/K+ ATPase pump. chondroitin sulfate proteoglycan ) core protein, Corneal endothelial cells in PBK/ABK have reduced cross-reacted with human, were produced in rabbits pump site density compared to normals.10 This func- (Couchman et al, manuscript in preparation). A tional impairment may be a secondary event in PBK/ monoclonal antibody to the aS chain of human type ABK. VI collagen (clone 3C4) was a gift from Dr. E. Engvall Several lines of evidence emphasize the impor- (Lajolla Cancer Research Foundation, Lajolla, CA). tance of ECM changes in PBK/ABK development: Polyclonal antibodies to human types I, III, and V collagen were obtained from Southern Biotechnology 1. Typically, PBK/ABK corneas develop ECM accu- (Birmingham, AL); polyclonal antibodies to human mulation at the endothelial (PCL) and the epi- 4 6 decorin and a monoclonal antibody to human tenas- thelial (SEF) levels. " cin (clone TN-2) were obtained from Chemicon Inter- 2. Corneal epithelial basement membrane (BM) in national (Temecula, CA); a monoclonal antibody to PBK/ABK lacks fibronectin, laminin, and type 14 human vimentin (clone Vim 3B4) was obtained from IV collagen, which might reduce cell adhesion Boehringer Mannheim (Indianapolis, IN); a mono- and facilitate bullae formation. clonal antibody to cellular fibronectin (clone IST-9) 3. PBK DM shows altered collagen content and lec- was obtained from Sera-Lab (Crawley Down, UK); a tin binding15 and, contrary to normal DM, does 13 monoclonal antibody to the al chain of bovine-hu- not support cell growth, suggesting structural man type VIII collagen (clone 9H3) was obtained from alterations, compositional alterations, or both. Seikagaku America (Rockville, MD); a polyclonal anti- Despite these findings, the ECM of PBK/ABK cor- body to human von Willebrand factor, a mixture of neas has not been studied systematically. monoclonal antibodies to human keratins (pankera- We report here on the detailed characterization tin cocktail), and a monoclonal antibody to human a- of the ECM and BM composition of PBK/ABK corneas smooth muscle actin (clone 1A4) were all obtained with antibodies to 27 individual ECM components. from Sigma Chemical (St. Louis, MO). Cross-species- Compared to normal corneas, there was an accumula- adsorbed fluorescein- and rhodamine-conjugated section of stromal tenascin and alterations of epithelial ondary antibodies were obtained from Chemicon In- BM and DM. We also describe the molecular and cellu- ternational. lar composition of PCL and SEF areas. Our results provide the documentation of significant fibrotic changes in PBK/ABK corneas on the molecular level, even in cases in which no PCL or SEF could be found. RESULTS We think PBK/ABK not only is a disease of chronic All PBK/ABK corneal buttons studied represented edema but has an ongoing fibrosis that plays an im- only the central corneal part, so their epithelial BM portant role in its pathology. structure was compared with that of normal central corneas only (see16 for differences of the epithelial BM MATERIALS AND METHODS composition in normal central cornea and limbus). In Normal adult human corneas (n = 14) were obtained PBK/ABK corneas and failed corneal grafts of pseu- within 36 hours of death from the National Disease dophakic-aphakic eyes (Table 1), similar ECM and

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TABLE l. PBK/ABK Patient History Time Between Cataract Removal and Corneal Patient Age Transplantation Visual Tenascin in Number (years) Sex (years) Diagnosis Acuity* Midstroma

1 75 F 2 PBK HM •+• 2 74 M 0.7f Failed graft, pseudophakic HM + 3 73 M 12f Failed graft, pseudophakic HM + 4 74 M 13f Failed graft, aphakic HM + 5 82 F 10 PBK CF -t- 6 69 M 3 PBK CF — 7 82 F 8 PBK CF — 8 69 F 1 PBK CF — 9 86 F 19J PBKt CF - 10 56 M 3 ABK CF — 11 79 M 5 ABK CF — 12 89 F 24 ABK CF — 13 80 F 6t Failed graft, aphakic CF + 14 83 M 12 PBK 20/150 — 15 81 F 4 PBK 200/400 — 16 57 F 12 PBK 20/50 - 17 57 M 8 PBK 20/60 - 18 79 F 3 PBK 20/400 - 19 68 M 6 PBK 200/100 - 20 78 M 10 PBK 20/150 — HM = patient recognizes hand movements; CF = patient can count fingers; + = present; — = absent; ± = local and/or weak expression. * 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 on stromal face (Fig. 2B). This was in contrast to normal these two groups were combined. age-matched corneas in which these chains usually were found on the stromal face of the DM (Fig. 2A). Tenascin In more than half of PBK/ABK corneas, the epithelial Normal central corneal epithelial BM, stroma, and BM was partially positive for these chains, a pattern DM were negative for tenascin (Figs. 1A, 1C). In con- that was never seen in normal central corneas (not trast, all PBK/ABK corneas had fibrillar tenascin de- shown). The distribution of the a$(TV) -a6(IV) chains posits in the anterior stroma beneath Bowman's layer was similar to that in normal corneas, with infrequent (Fig. IB) and in the posterior stroma adjacent to the local discontinuities of the epithelial BM (not shown). DM (Fig. ID). Tenascin also was present in parts of Using a new panel of monoclonal antibodies to 18 epithelial BM and the DM (not shown), in areas of all six a (IV) chains, we have expanded our recent 16 bullae formation (Fig. IB), SEF, and PCL (Fig. ID). In results on the heterogeneity of a (IV) chain distribu- some cases, usually more advanced (Table 1), tenascin tion in the epithelial BM of central cornea, limbus, deposits were seen in the midstroma as well. It should and conjunctiva. Both the a5(IV) and the a6(IV) be noted that in normal limbus, tenascin was found chains were found in the central, limbal, and conjunc- in the stroma and only in a part of limbal blood vessels tival [weaker a5(W) staining than in the limbus] re- (not shown). This vessel staining pattern is similar to gions of epithelial BM. Staining for a3(IV) and a4(IV) that observed in other tissues for tenascin-C but differs chains was continuous in the central cornea and discontinuous or absent in the limbus and proximal con- from that of another tenascin variant, tenascin-X, al- 16 ways associated with blood vessels.20 These data sug- junctiva, but it could reappear in distal conjunctiva gest that our antibody recognized the ubiquitous ten- on sections denatured in 6 M urea. In the DM, a6(IV) ascin variant, tenascin-C. chain was seen on the endothelial face, as were a3(TV)-a5(W) chains. Unlike the latter three chains, Type IV Collagen Chains a6(IV) chain was not found around keratocytes. In all PBK/ABK corneas, the staining for al(IV) and Fibronectin a2(IV) chains was observed on the endothelial face The PBK/ABK corneas had local fibrillar accumula- of the DM, often together with the staining of the tions of fibronectin (including cellular isoform) in the

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FIGURE l. Tenascin in normal and PBK/ABK corneas, No staining is seen in normal corneas in the anterior (A) and posterior (C) parts. In PBK/ABK corneas, tenascin fibrils are found in the anterior stroma and beneath epithelial cells in a bulla (B), as well as in the posterior stroma, on the endothelial face of the DM, and in die PCL

anterior stroma (Table 2). The stromal DM face was negative, but the endothelial face was positive (Fig. 2D), unlike the normals that displayed a reverse staining pattern (Fig. 2G). The PBK/ABK epithelial BM FIGURE 2. DM and epidielial BM alterations in PBK/ABK cor- showed discontinuous staining (not shown); SEF areas neas. (AJB)> type IV collagen (staining with an antibody to the (Fig. 4F) and PCL (Fig. 2D) were positive. al—«2 triple helical domain); (C,D), fibronectin; (E,F), laminin (31 (Bl) chain in normal (A.C.E) and diseased (B,D,F) corneas. Laminin Chains Note die appearance of al—a2 type IV collagen (B) and fibronectin (D) on the endodielial face of the DM and in the PCL, The PBK/ABK corneas (Table 2) displayed much as well as a weak, discontinuous staining of the epithelial BM weaker than normal and often discontinuous staining for laminin /?1 chain (F) in PBK/ABK corneas. Fibronectin of the epithelial BM for laminin-1 (al/31yl, or A-Bl- disappears from the stromal face of the DM in diseased corneas B2) chains (Figs. 2E, 2F). In contrast to this, laminin- (compare D,C). E = epithelium; S = stroma; B = Bowman's 5 (kalinin-nicein) was preserved in the epithelial BM layer; P = posterior collagenous layer. Bar = 40 /Am. of most PBK/ABK corneas, as was another anchoring fibril protein, type VII collagen (not shown). Sparse deposits of laminin-1 were seen in SEF areas (Fig. 4E) Entactin/Nidogen and in the PCL (not shown). In the DM, laminin-1 Entactin/nidogen staining in PBK/ABK (not shown) had normal distribution. Laminin chains a2 (M) and was similar to that for laminin-1—that is, it was discon- /?2 (S), normally absent from central corneal epithe- tinuous and often absent from the epithelial BM and lial BM,16 did not appear in PBK/ABK corneas (not remained normal in the DM (Table 2). SEF areas and shown). PCL (Fig. 5C) contained some entactin/nidogen.

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TABLE 2. Major ECM Abnormalities in PBK/ABK Corneas ECM Subepithelial Component Epithelial BM Stromal ECM DM Fibrosis PCL

Tenascin Partially positive Anterior and Positive Positive Positive posterior depositsf al-a2 type IV Partially positive Anterior deposits Endothelial face Positive Positive collagen positive Fibronectin Partially negative Anterior deposits Stromal face Positive Positive mostly negative Laminin-1 Partially negative No change* No change* Mostly negative Weakly positive Entactin/ Partially negative No change* No change* Mostly negative Weakly positive nidogen Bamacan No change* No change* No change* Weakly positive Weakly positive Decorin No change* No change* No change* Positive Positive Type VIII Occasionally No change* in Endothelial face Positive Positive collagen positive most cases positive Type XIV No change* No change* No change* Positive Positive collagen ECM = extracellular matrix; BM = basement membrane; DM = Descemet's membrane; PCL = posterior collagenous layer. * Compared with normal corneas f In some more advanced cases, tenascin deposits in midstroma were also found (see Table 1).

Bamacan the endothelial face of the DM (Fig. 3E), and less This proteoglycan, revealed by the core protein anti- intensely in Bowman's layer. In diseased corneas, it body, was found around keratocytes, endothelial cells was seen in SEF areas and in PCL (Figs. 3F, 5E) with- (Fig. 3A), and limbal blood vessels in the normal cor- out changes in the stroma. neas. This was unusual because in other tissues, bama- Type XIV Collagen can exclusively associated with BMs.19 In PBK/ABK corneas, it also was seen on the endothelial face of Only weak and irregular staining around keratocytes the DM and in the PCL (Fig. 3B) and was associated was observed in normal central corneas. In the limbus, with SEF, including the BM of abnormal epithelium stroma, blood vessels, and epithelial BM were positive (Fig. 4D). (not shown). In PBK/ABK corneas, however, this collagen was accumulated in SEF areas (not shown) and Decorin in PCL (Fig. 5B). As in rabbit corneas,21 decorin core protein was dis- tributed uniformly in the stroma of normal human Correlation Between Clinical Features and corneas (Fig. 3C) and in Bowman's layer. In diseased Extracellular Matrix Changes corneas, it was seen on the endothelial face of the Analysis of case histories showed no correlation be- DM, in the PCL (Fig. 3D), and in SEF areas (not tween the disease duration and severity (evaluated as shown). The stromal pattern did not change in PBK/ a degree of vision loss). As shown in Table 1, patients ABK (see also3). could be arbitrarily divided into three groups by their best-corrected visual acuity before corneal transplanta- Type VIII Collagen tion: recognizing hand movements (severe vision In normal central corneas, it was localized at the stro- loss), counting fingers, and having visual acuity of 20/ mal face of the DM (see also22) and as weakly stained 400 or better. For most of the ECM changes (including fine fibrils in the anterior stroma. Limbal stroma and the presence and cellularity of SEF and/or PCL), some of the blood vessels were positive (not shown). there was no correlation with visual acuity decrease. In the diseased corneas, type VIII collagen was seen However, the presence of tenascin in midstroma (Ta- at the endothelial face of the DM, in SEF areas (not ble 1) was significantly more frequent in group 1 (4 shown), and in PCL (Fig. 5A). Stromal distribution of 4) compared to group 2 (2 of 9; P = 0.021) and usually did not change (not shown). especially to group 3 (0 of 7; P = 0.003). There was also a tendency, though not significant, to an in- Type XII Collagen creased incidence of tenascin in midstroma in group In normal corneas, type XII collagen was found at the 2 compared to group 3. The presence of tenascin in level of the epithelial BM, throughout the stroma, on midstroma correlated with visual acuity decrease and

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Bowman's layer was noted previously in PBK/ABK corneas.'1'715 In half the PBK/ABK corneas studied here, abnormal ECM deposits with or without cells were found between the epithelium and the Bowman's layer. Two types of these subepithelial deposits were distinguished here. The first one may be termed ECM- type deposit because only ECM without cells was present between the epithelium and the Bowman's layer (Fig. 4A). This type of SEF had abnormal accumula- tions of fibronectin (including cellular), tenascin, perlecan, decorin, and types I, III, IV (mostly al-a2 chains), V, VI, VIII, XII, and XTV collagen, with some laminin-1, laminin-5, entactin/nidogen, and bamacan (Figs. 4B to 4E). Staining for stromal components was more pronounced than for most BM components (see Figs. 4B to 4E). The second type of SEF (fibrocellular type) contained cells embedded in the ECM, which was 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 myofibroblasts, but did not stain for keratins (not shown here). These cells may be the actual source of the abnormal subepithelial ECM. In areas of bullae without SEF, the basal surface of detaching epithelium also stained for abnormal ECM (Fig. IB). Some ECM accumulated between epithelial cells or layers (Figs. IB, 4D, 4F) in contrast to the normal corneas. Posterior Collagenous Layer Composition of PBK/ABK Corneas The PCL often is found in PBK/ABK corneas as an additional fibrous layer between die remnant DM and the endothelial cells. In our series of PBK/ABK, the PCL was observed in 80% of cases, in accordance with previous studies.615 PCL contained types VI, XII, and XIV collagen, tenascin, fibronectin, decorin, bama- FIGURE3. Bamacan (A,B). decorin (C,D), and type XII colla- can, perlecan, some laminin-1 and entactin-nidogen gen (E,F) in normal (A,C,E) and diseased (B,D,F) corneas. (Figs. ID, 2D, 3B, 3D, 3F, 5A to 5E), in addition to Note abnormal deposition of bamacan (B), decorin (D), the previously reported types I, III, IV, V, and VIII and type XII collagen (F) on the endothelial face of the collagen.1523'24 The presence of tenascin, type XTV col- DM and in the PCL in PBK/ABK corneas. E = epithelium; lagen, bamacan, and decorin may be significant be- S = stroma; P = posterior collagenous layer. Bar = 40 jj,m. cause they were not seen in normal DM but appeared in diseased DM and in the PCL. PCL also contained al-a2 type IV collagen (together with some aS~a6 may reflect a more advanced fibrosis. Midstromal ten- chains) and fibronectin, including cellular isoform ascin was also a common finding in failed grafts (4 of (Fig. 2D), that were not observed normally on the 4), compared to primary PBK/ABK (2 of 16; P = endothelial face of the DM.16 Thus, PCL was an accu- 0.003; Table 1). mulation of corneal stromal and BM components. Extracellular Matrix Composition of This ECM-type of PCL resembled the ECM-type of Subepithelial Fibrosis Areas and Bullae in SEF. In two cases, well-developed PCLs with myofi- PBK/ABK Corneas broblasts inside them (Figs. 5D to 5F) were found (see also25), apparently corresponding to the fibrocellular- Bullae are regions of fluid accumulation in PBK/ABK type SEF. corneas either beneath the epithelium or between the epithelial cells. After the bullae resorb, ECM may be DISCUSSION deposited in these areas. A subepithelial fibrocellular PBK/ABK remains a leading indication for corneal material (pannus) disrupting the epithelial BM and transplantation, but there is litde basic information

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FIGURE 4. Subepithelial fibrosis in PBK/ABK corneas. (A) Hematoxylin staining of an area with extracellular matrix-type SEF, No abnormal cells are seen between the epithelium and the 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. Note heavy deposits of stromal collagens type V and XII compared to scarce deposits of BM components (bamacan and laminin-1). (F to H) Fibrocellular-type SEF with cellular fibronectin 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 theory mal fibrotic deposition of ECM proteins was detected that intraocular lenses affect the protein levels or com- even in PBK/ABK corneas without evidence of SEF or plement factor C3 or C3a in aqueous humor that could PCL formation. The most conspicuous alteration of play a role in inflammation. The stroma of the PBK/ ECM pattern in PBK/ABK corneas was an induction ABK corneas has decreased keratan sulfate content,3'27 of stromal tenascin. but the distribution of types V and VI collagen does Tenascin is a large hexameric glycoprotein com- not change.28 One study described lack of laminin, posed of multiple domains.30"33 The tenascin family fibronectin, and type IV collagen in the epithelial BM comprises three proteins that are different gene prod- of PBK/ABK corneas.14 PBK/ABK corneas often have ucts3233: tenascin-C (the first discovered), tenascin-R a thickened DM with an accumulation of new fibriUar (restrictin), and tenascin-X (X gene product). They ECM with or without cells (PCL) between the endo- may play a role in cell migration and proliferation, thelium and the DM.23'25'29 This ECM contains types I tissue repair, and oncogenesis.30'31'33 In addition, alter- and V collagen,24 as well as some type III collagen,23 native splicing of fibronectin type Ill-like (FN-HI) re- that are not present in this location in normal corneas. peats generates a number of isoforms.34 Tenascin is Here, the evidence for ongoing fibrosis in PBK/ antiadhesive. Cells may attach to it but do not spread ABK corneas is provided. In 16 of 20 cases, a fibrotic well.35 The cell-binding sites are in the FN-III domains PCL containing stromal ECM and BM components II-VI and in the fibrinogen-like domain, whereas the was seen, and in 10 of 20 cases, SEF with ECM deposi- epidermal growth factor-like domain is antiadhe- tion was observed. It should be noted that the abnor- sive.35"37 Tenascin can weaken cell-substrate attach-

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it appears near the wound edges after fibronectin and disappears more slowly than fibronectin.45"47 Its cellular origin in the wounded corneas is unknown. In all PBK/ABK corneas studied here, abnormal tenascin deposits 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 advanced cases, tenascin also appeared in midstroma, which correlated with loss of visual acuity (Table 1). In all but one of the studied corneas, the time between cataract surgery and corneal transplantation for subsequent PBK/ABK varied from 1 to 24 years (Table 1). In rabbit corneal wound healing, tenascin can be detected in the scar area for only as long as 1 year after injury.45"47 These data suggest that tenascin accumulation in PBK/ABK corneas probably is not part of corneal wound healing associated with cataract surgery and is more likely triggered later in the course of the disease. Tenascin may be upregulated in PBK/ABK by the following mechanism. In the diseased corneas, stromal fluid accumulation and a resultant swelling may create a significant mechanical stress to stromal keratocytes. In this situation, the cells could attempt to relieve the strain and preserve normal stromal structure by contracting their surrounding collagen. In cultured fibroblasts, such collagen contraction has been shown specifically to induce production and deposition of tenascin.48 It is thus possible that tenascin induction in PBK/ABK corneas could be triggered by mechanical stress. Some data seem to support this assumption. The degree of edema in PBK/ABK-failed corneal grafts is higher dian in primary PBK/ABK49 Ac- cording to the stress hypothesis, the failed grafts should have more stromal tenascin tfian primary PBK/ABK corneas. Indeed, all four failed grafts studied here showed marked staining for tenascin in midstroma, in addition to anterior and posterior stroma. However, of 16 corneas widi primary PBK/ ABK, staining for tenascin in midstroma was noted FIGURE 5. Posterior collagenous layer in PBK/ABK corneas. only in two, and it was weaker than in failed grafts (A to C) Extracellular matrix-type PCL containing types VIII (Table 1). (A) and XIV (B) collagen and entactin/nidogen (C). (D to Another salient feature of PBK/ABK corneas was F) Fibrocellular-type PCL containing types VI (D) and XII an alteration of BM structure. In accordance with pre- collagen (E) and numerous myofibroblasts positive for a- vious data,14 there was a reduction (up to a complete smooth muscle actin (F). S = stroma; P = posterior collage- absence) of fibronectin, laminin-1, and entactin-ni- nous layer. Bar = 40 jxm. dogen in the epithelial BM. This could lead to a weaker epithelial adhesion to the Bowman's layer and ment and promote cell detachment by loss of intercel- could facilitate bullae formation. The DM alterations lular contacts and downregulation of focal adhe- in PBK/ABK corneas were also found. They primarily sions.303338'39 Its expression can be induced by concerned al-a2 type IV collagen and fibronectin. fibrogenic cytokines,40"42 such as transforming growth In normal-aged corneas, both proteins usually were factor-/3l, basic fibroblast growdi factor, and platelet- seen on the stromal face of the DM.16 However, in derived growth factor-BB. PBK/ABK corneas, they were found on the endothe- In normal cornea, tenascin is present only in the lial face of the DM, irrespective of the presence or limbal stroma and BM.43M In corneal wound healing, absence of a PCL (Figs. 2B, 2D; Table 2)/It is unclear

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which cells are responsible for these changes in PBK/ decrease in visual acuity of patients and the appear- ABK. They could be keratocytes, which are presumably ance of tenascin in midstroma that may be a marker the normal source of fibronectin and of al-a2 type of advanced fibrosis. The major ECM abnormalities IV collagen in the DM. On the other hand, endothelial found in PBK/ABK corneas cannot be explained eas- cells, which apparently do not deposit these proteins ily by an altered fluid balance. Vision-threatening in normal corneas,16 could acquire an altered ECM changes may result from abnormal production and pattern during PBK/ABK development. It is notewor- deposition of ECM by dysfunctional cells, which may thy that cultured corneal endothelium responded to exacerbate the disease. By inference from other sys- the endogenous basic fibroblast growth factor by mod- tems, excessive ECM production in PBK/ABK corneas ulating ECM production from type IV to types I and could be modulated by fibrogenic cytokines, trans- V collagen.50'51 Because basic fibroblast growth factor forming growth factor-/?l(— /32), basic fibroblast can induce fibrosis, it might contribute to the changes growth factor, or platelet-derived growth factor. Direc- of the DM composition in PBK/ABK. tional modulation of the levels of involved cytokines A common feature of PBK/ABK corneas is the may make it possible to normalize corneal ECM and formation of PCL and of bullae with subsequent SEF. to slow down or stop the disease progression, as dem- 53 This article provides a detailed description of the ECM onstrated for fibrotic diseases in other organs. Only molecular composition in these areas. In both cases, then, transplantation of normal endothelium to re- abnormal ECM had a similar composition and was a store its function could become feasible. mixture of stromal (tenascin, decorin, and collagen types I, III, V, VI, VIII, XII, and XIV) and BM compo- Key Words nents (type IV collagen, laminin-1, entactin-nidogen, basement membrane, bullous keratopathy, corneal edema, perlecan, bamacan, and fibronectin). Importantly, extracellular matrix, immunocytochemistry PCL contained tenascin in all cases. This antiadhesive glycoprotein could provide an ECM environment for Acknowledgments the endothelial cells in which they would be unable to attach normally, to spread, and to maintain normal The authors thank Drs. T. Perl (Corneal Associates of New corneal hydration (see also13). Studies are under way Jersey, West Orange, NJ) for providing tissue, E. Engvall to determine which cells produce tenascin in the (La Jolla Cancer Research Foundation, La Jolla, CA) for PBK/ABK corneas, which cytokines trigger its induc- antilaminin antibodies, A. F. Michael (University of Minne- tion, and which tenascin splice variants are expressed. sota, Minneapolis, MN) for anti-a5(IV) antibody, D. J. Brown (Cedars-Sinai Medical Center, Los Angeles, CA) for Staining for stromal components was considerably valuable comments, and Z. Huang for expert assistance. An- stronger in PCL and in SEF than for most BM compo- tibodies to laminin (52 chain, produced by Dr. J. Sanes, and nents (see, for example, Fig. 4), suggesting that cells to type IV collagen al-a2 chains, produced by Dr. H. that normally produce stromal ECM (apparently kera- Furthmayr, were obtained from the Developmental Studies tocytes) may contribute to its abnormal accumulation Hybridoma Bank, Department of Biology, University of Iowa in PBK/ABK corneas. 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