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 cor- neas 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 epithe- lial, 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 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- 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 Investigative Ophthalmology & Visual Science, May 1996, Vol. 37, No. 6 Copyright © Association for Research in Vision and Ophthalmology 997 Downloaded from iovs.arvojournals.org on 09/27/2021 998 Investigative Ophthalmology & Visual Science, May 1996, Vol. 37, No. 6 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 sec- tion 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