ARTICLE |
Correspondence to: Alexander V. Ljubimov, Ophthalmology Research Laboratories, CedarsSinai Medical Center, 8700 Beverly Boulevard, Davis-5069, Los Angeles, CA 90048. E-mail: ljubimov@cshs.org
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Summary |
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We previously found an abnormal deposition of an extracellular matrix glycoprotein, tenascin-C (TN-C), in human corneas with pseudophakic/aphakic bullous keratopathy (PBK/ABK). In this work, we studied cellular TN-C receptors in normal and PBK/ABK corneas. Cryostat sections of normal and PBK/ABK corneas were stained by immuno-fluorescence for TN-C receptors: 2,
8,
9,
Vß3, ß1, and ß6 integrins, and annexin II. ß6 integrin mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) using ß2-microglobulin gene to normalize the samples. In PBK/ABK compared to normal corneas, relatively minor changes were observed for
2 and ß1 integrins, and for annexin II.
8,
9, and ß6 subunits of TN-C receptors,
8ß1
9ß1, and
Vß6, respectively, were absent from normal central corneas but were found in the central epithelium of PBK/ABK corneas. ß6 integrin showed the most significant accumulation. It correlated best with the expression of TN-C rather than with the expression of other
Vß6 ligands, fibronectin, and vitronectin. RT-PCR analysis also showed elevated levels of ß6 mRNA in PBK/ABK compared to normal corneas. Therefore, accumulation of TN-C in PBK/ABK corneas was accompanied by an increased expression of its three binding integrins, especially
Vß6 in the corneal epithelium. The interaction of tenascin-C with these integrins may contribute to the fibrotic process that occurs in PBK/ABK corneas.
(J Histochem Cytochem 49:13411350, 2001)
Key Words:
bullous keratopathy, corneal epithelium, tenascin-C, Vß6 integrin,
8 integrin,
9 integrin, annexin II, RT-PCR, immunofluorescence
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Introduction |
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PSEUDOPHAKIC (PBK) and aphakic (ABK) bullous keratopathy is a common blistering corneal disease and is the leading indication for corneal transplantation and regraft (
We have previously shown that PBK/ABK corneas presented signs of an ongoing fibrotic process characterized by excessive deposition of specific extracellular matrix (ECM) components (
TN-C may be adhesive for some cell types and antiadhesive for others (2ß1,
8ß1
9ß1,
Vß3, and
Vß6 (
8ß1,
9ß1,
Vß6) appeared in PBK/ABK corneas. Of these integrins,
Vß6 integrin showed the most significant correlation with TN-C overexpression.
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Materials and Methods |
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Tissue
A total of 15 normal and 38 age-matched PBK/ABK corneas were used. Diseased corneas removed during corneal transplantation were received from local collaborating surgeons. Tissue collection was conducted under the CedarsSinai Medical Center IRB protocol number 3201. Normal autopsy corneas were received from the National Disease Research Interchange (Philadelphia, PA), which has a human tissue collection protocol approved by a managerial committee and subject to National Institutes of Health oversight. All tissues were processed within 30 hr after death or corneal transplantation. For PBK/ABK corneas, only a central part was available that had been removed during transplantation. Because of this, all ECM abnormalities in PBK/ABK corneas were previously documented in the central part only, where Bowman's layer lies underneath the epithelium (
Immunohistochemistry
Corneas (10 normal and 26 age-matched PBK/ABK) were bisected and embedded in OCT compound (Ted Pella; Redding, CA). Six-µm cryostat sections were prepared and stained by indirect immunofluorescence for ECM components and integrins, as previously described (Vß3 (clone LM609),
3ß1 (clone M-KID 2),
1 I domain (clone FB12),
6 (clone NKI-GoH3), ß1 (clone HB1.1), ß4 (clone 3E1) (all from Chemicon International; Temecula, CA), and ß6 (clone E7P6;
5ß1,
2 (all from Chemicon International),
8 (
9 (
9 reacted in an identical way. Cross-species adsorbed fluorescein- and rhodamine-conjugated secondary antibodies were from Chemicon International. In double-label experiments (not shown here), it was found that in PBK/ABK corneas, affinity-purified polyclonal antibody to TN-C (Chemicon International) reacted identically with the monoclonal antibody BC-8.
Semiquantitative RT-PCR
This was done as previously described (
PCR was carried out with 25 ng of reverse-transcribed RNA, using 1.25 U Taq polymerase (Invitrogen Life Technologies) and 2 mM Mg++. Each cycle consisted of 30-sec denaturation at 94C, 30-sec annealing at 65C, and 45-sec elongation at 72C. Samples normalized by ß2-MG amplification were amplified in a linear range established using serial cDNA dilutions and varying the number of cycles. A total of 33 cycles were used for ß6 integrin and 31 cycles for ß2-MG. Amplified products were separated by electrophoresis in 3% agarose gels and visualized under UV light after staining with ethidium bromide. Routine RT-PCR controls without reverse transcriptase were negative.
Southern blotting analysis was done as described previously (
Statistical Analysis
Immunostaining data were analyzed by the two-sided Fisher's exact test using InStat software program (GraphPad Software; San Diego, CA). To this end, the number of cases with abnormal staining pattern in one experimental group (e.g., normal) was compared to the number of cases with abnormal staining pattern in another experimental group (e.g., PBK/ABK). RT-PCR band relative intensities were compared by the two-tailed unpaired Student's t-test using the InStat program and were expressed as mean ± SEM. With both methods, p<0.05 was considered significant.
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Results |
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Appearance of Tenascin-C-binding Integrins 8ß1,
9ß1, and
Vß6 in PBK/ABK corneas
Normal central corneal epithelial cells and keratocytes expressed the TN-C receptors 2ß1 integrin and annexin II, as determined by immunofluorescence (Fig 1). In the corneal epithelium, distinct basal cell surface staining was seen for both components. In central PBK/ABK corneas, these receptors were not changed in about half of cases. In the remaining cases (5/11),
2 integrin appeared reduced in the epithelium (Fig 1). In 6/11 PBK/ABK cases, more (Fig 1) or less staining than normal was observed for annexin II, especially at the basal surface of basal epithelial cells. The stromal staining for both these receptors did not change in diseased corneas.
Vß3 integrin was not found either in normal or in PBK/ABK corneas (Fig 1), in accordance with previous data (
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Subunits of the TN-C-binding integrins 8 and ß6 were absent in normal corneas, both in the central part (Fig 2, upper row) and the limbus (not shown). Only
9 integrin, in accordance with previous data in mice (
8 integrin, 11/17 (65%) were positive for
9, and 12/15 (80%) were positive for ß6. All these integrins were mostly expressed in basal epithelial cells, whereas stromal keratocytes and endothelial cells were negative (Fig 2). The expression of these integrins was heterogeneous. Some PBK/ABK cases showed small local areas of positive staining, whereas in other cases the staining for these integrins was observed throughout large parts of the basal epithelium up to a whole cornea. The induction of these integrins was next correlated with the subepithelial expression of TN-C. For
8 integrin, such correlation was found in 8/17 cases (47%), for
9 in 9/17 cases (53%), and for ß6 in 11/15 cases (73%).
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TN-C-binding ß6 integrin, the most increased in PBK/ABK corneal epithelium, also binds to fibronectin and vitronectin. In a separate set of experiments, we tested by double immunofluorescence on serial sections whether ß6 expression in these corneas correlated with the expression of either vitronectin or fibronectin or TN-C. The correlation was judged to be positive both in cases where the integrin and the ligand were present (the integrin, in the epithelium, and the ligand, in the subepithelial region and/or epithelial BM), and in cases where the integrin and the ligand were both absent. Double staining for fibronectin and ß6 integrin, and also for vitronectin and ß6 integrin, was done using pairs of mouse monoclonal antibodies together. This was possible because the patterns of the ligands and the receptor did not overlap. Fibronectin and vitronectin were normally present in keratocytes (fibronectin also in the epithelial BM) but not in the epithelial cells, whereas ß6 integrin could be found only in the epithelial cells.
The results showed that ß6 expression in PBK/ABK corneal epithelium correlated with the expression of subepithelial fibronectin in 8/21 cases. Correlation of ß6 with vitronectin expression was seen in 7/20 cases. The correlation of ß6 with TN-C expression was significantly higher than with fibronectin or vitronectin (p<0.0008) and was observed in 21/24 cases (three of these cases were negative for both ß6 and subepithelial TN-C) (Fig 3). These results suggested that ß6 integrin appeared coordinately with TN-C in PBK/ABK corneas.
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We have previously shown that PBK/ABK corneas also displayed a decreased and discontinuous staining of the epithelial BM for fibronectin, Type IV collagen, laminin, and nidogen-1/entactin (5ß1 (fibronectin receptor),
6ß4 (laminin receptor), and
3ß1 (collagen/laminin/entactin receptor) did not change in PBK/ABK epithelium (Table 1).
1ß1 (collagen receptor) was not found in any of the central corneas (not shown). These results suggest that epithelial cell integrin changes in PBK/ABK may reflect the abnormal presence of TN-C rather than alterations of major epithelial BM components or nonspecific corneal damage during storage and handling.
Increased Expression of ß6 Integrin mRNA in PBK/ABK Corneas
Semiquantitative RT-PCR from total corneal RNA demonstrated that normal corneas had relatively low levels of ß6 integrin mRNA that significantly increased in PBK/ABK corneas (Fig 4). The average increase of ß6 integrin mRNA expression in PBK/ABK vs normal corneas was approximately twofold and statistically significant (p<0.008). No correlation was found between ß6 integrin mRNA levels and the degree of visual acuity loss in PBK/ABK corneas examined before corneal transplantation.
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Discussion |
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Tenascins form a family of five related large, hexameric ECM glycoproteins with modular structure. They play a role in various cell functions, including adhesion, migration, differentiation, and proliferation. In the extracellular space, tenascins can bind a plethora of other ECM molecules. Tenascins also bind to the surface of many cell types using several defined receptors, most of them belonging to the integrin family (reviewed in
TN-C is fairly abundant in normal central fetal corneas (
We hypothesized that abnormal expression of TN-C in PBK/ABK corneas might be accompanied by altered expression of some of its binding proteins. A systematic study of these proteins in normal and PBK/ABK corneas was conducted. We found some TN-C receptors in both normal and PBK/ABK corneas, including 2ß1 integrin and annexin II. These receptors showed abnormal staining in about half of diseased corneas. The
2ß1 integrin is also a laminin/collagen receptor and was usually decreased in PBK/ABK epithelium. This might be due to laminin and/or collagen alterations seen in the epithelial BM of PBK/ABK corneas (
2ß1 integrin and annexin II were not further studied.
Three TN-C-binding epithelial integrins, 8ß1,
9ß1, and
Vß6, did show correlation with TN-C expression. All of them, like TN-C, were only found in PBK/ABK central corneas. The corneal
9ß1 integrin has been thoroughly studied by Stepp's group (
8ß1 integrin distribution in normal and PBK/ABK human corneas, which was largely similar to that of
Vß6 integrin.
The 8ß1 integrin can serve as an RGD-dependent receptor for TN-C, fibronectin, vitronectin, and osteopontin (
8ß1 could only bind to TN-C fragments but not to total protein (
The 9ß1 integrin is expressed in epithelial, muscle, and neutrophil cells and binds to vascular cell adhesion molecule 1 (VCAM-1), TN-C, osteopontin, transglutaminase, and other ligands (
9ß1 integrin (
9ß1 integrin may interact with TN-C. These two proteins co-distribute during oral mucosa wound healing and in proliferative disorders (
9ß1 integrin is confined to the limbal basal epithelium (
9ß1 integrin is also induced in the central corneal epithelium after wound healing in TN-C knockout mice (
The Vß6 is an epithelial integrin and can bind TN-C, fibronectin, and vitronectin (
Vß6 integrin (
As we showed here, Vß6 integrin was absent from normal corneal epithelium but became expressed in most PBK/ABK corneas. Increased mRNA levels accompanied its appearance in diseased corneas. An important finding was that
Vß6 integrin expression specifically correlated with the deposition of subepithelial TN-C. At the protein level, such a correlation was also observed at late stages of oral mucosal healing (
Vß6 integrin may be first expressed in PBK/ABK corneal epithelium as a result of epithelial alterations including edema, remodeling, and proliferation. This may activate corneal TGF-ß which, in turn, could induce the expression of subepithelial TN-C as part of the fibrotic process typical for this corneal disorder. Alternatively, TN-C may be upregulated by IGF-I elevated in PBK/ABK corneas (
Vß6 integrin appearance in the epithelium.
TN-C is associated with inflammation and wound healing in remodeling and pathological tissues (9ß1 integrin is still induced during corneal wound healing, suggesting that it binds to a different ligand (
Vß6 in TN-C knockout mice, but mouse-reacting antibodies to this integrin that would stain tissue or work in Western blotting are not available.
At the same time, TN-C knockout mice have abnormal wound healing in corneal stroma (
In summary, PBK/ABK corneas show abnormal upregulation of several TN-C-binding epithelial integrins, including Vß6. The epithelial expression of this integrin spatially correlates with that of subepithelial TN-C (but not fibronectin or vitronectin) in diseased corneas and may be part of and/or contribute to the fibrotic process going on in PBK/ABK corneas.
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Footnotes |
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Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology (ARVO), Fort Lauderdale, FL, May 1998.
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Acknowledgments |
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Supported by grants from the NIH (EY10836 to MCK), the Braille Transcribers Guild (AVL), the Discovery Fund for Eye Research, and the Skirball Program for Molecular Ophthalmology (AVL, MCK).
We thank Drs Anthony Nesburn, Ezra Maguen, Ronald Gaster, Wayne Bowman, John Hofbauer, Theodore Perl, and Ira Wong for providing us with bullous keratopathy corneas, and the National Disease Research Interchange for supplying normal human corneas. We are grateful to Drs Mary Ann Stepp (George Washington University Medical Center; Washington, DC) and Luciano Zardi (Istituto Nazionale per la Ricerca sul Cancro; Genoa, Italy) for donating antibodies.
Received for publication March 6, 2001; accepted June 4, 2001.
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