Abnormal extracellular matrix in corneas with pseudophakic bullous keratopathy.

The present study describes biochemical and morphological differences of pseudophakic bullous keratopathy (PBK) corneas as compared with normal corneas. At the ultrastructural level, all PBK corneas studied had abnormal fibrillar material posterior to the Descemet's membrane. In addition, two of the six PBK buttons had subepithelial fibrocellular materials disrupting the epithelial basement membrane and Bowman's layer. Aggregates of collagen fibrils with 110 nm periodicity were occasionally seen within the stroma of the PBK corneas. Isolation and purification of the collagen from the Descemet's membrane/posterior collagenous layer (DM/PCL) showed an increased amount of material with molecular weight in the range of 50-60K daltons (presumably type VIII collagen) and decreased amounts of higher molecular weight, disulfide-bonded collagenous materials (presumably type IV collagen) as compared with normals. Sugar-specific lectin studies showed an increased deposition of peanut agglutinin (PNA) and Ricinus communis agglutinin I (RCA120) in the DM/PCL of the PBK corneas. Our data suggest that the DM/PCL of PBK corneas have an increased accumulation of terminal B-galactose and B-D-galactose (1-3)-D-N-acetylgalactosamine residues and altered ratios of low and high molecular weight collagenous proteins.

Characterization of stroma from Fuchs' endothelial dystrophy corneas.

Fuchs' endothelial dystrophy is commonly regarded as an endothelial cell disorder. In the present study we compared glycoconjugates of Fuchs' and normal corneas using FITC conjugated lectins [peanut agglutinin (PNA), castor bean agglutinin (RCA120), soybean agglutinin (SBA), and wheat germ agglutinin (WGA)]. Our results showed increased staining with RCA120 and PNA in the posterior region of the Fuchs' corneas, indicating an accumulation of terminal beta-galactose and B-D-galactose (1-3)-D-N-acetylgalactosamine residues. The stromal and epithelial regions of normal and Fuchs' corneas exhibited similar staining patterns with all lectins tested. Our collagen studies showed an increased extractability and abnormal amino acid analyses of collagen from Fuchs' corneas as compared with normals. The purified collagens did have similar banding patterns by sodium dodecyl sulfate gels. However, further characterization by 125(1) two-dimensional peptide mapping revealed that Fuchs' alpha 1-sized chains contained fingerprints that were distinctly different from normal cornea stromal collagen. These data suggest that in addition to abnormal accumulation of RCA120- and PNA-specific glycoconjugates in the posterior cornea, Fuchs' corneas contained stromal collagens with altered biochemical properties. We postulate that the characteristic deterioration of endothelial function in Fuchs' dystrophy may compromise the microenvironment of the stroma and its keratocytes, and thereby lead to an altered collagenous extracellular matrix.

Altered gelatinolytic activities in an apparent unilateral keratoconus patient. A case report.

Recent studies have suggested that one possible cause of keratoconus involves increased degradation of the corneal extracellular matrix. Most studies have examined corneas from patients with bilateral, sporadic keratoconus. In this report, both corneas from a 70-year-old man with a history of familial keratoconus and clinical signs of unilateral keratoconus were examined for gelatinase activity. Our results demonstrated a qualitative (three additional activity bands of 88, 92, and 100 kDa molecular weights) and quantitative differences (an apparent increase in overall amounts) in the gelatinolytic enzyme profiles of the affected keratoconus cornea as compared with his unaffected (nonkeratoconus) cornea, normal corneas, bilateral sporadic keratoconus corneas, and cultured keratocytes. In addition, gelatinolytic enzymes were more readily extracted from the affected cornea compared with controls. These data lend support to the hypothesis of a heterogeneous etiology for keratoconus and to previous suggestions that this disorder may be related to an alteration in extracellular matrix degradation.

Increased gelatinolytic activity in keratoconus keratocyte cultures. A correlation to an altered matrix metalloproteinase-2/tissue inhibitor of metalloproteinase ratio.

Keratoconus is a noninflammatory corneal disorder characterized by gradual stromal thinning and astigmatism. Altered degradation of corneal extracellular matrix is a suggested etiology for this disorder. In the present study we established keratocyte cultures from normal and keratoconus corneas and investigated the roles that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP, TIMP-2) may play. After chemical modification (reduction and alkylation) to remove the inhibitor and activation of enzyme with p-aminophenylmercuric acetate (APMA), keratoconus-conditioned media displayed a significant increase (p < 0.05) in the total potential gelatinolytic activity when compared with normal culture media treated in a similar manner. Basal levels of gelatinolytic activity in keratoconus culture media (no reduction, alkylation, or APMA treatment), determined by two different assay methods, tended to be about twice that of normal cell cultures. By zymography, both keratoconus and normal cultures showed identical enzyme patterns, which represented MMP-2 (72 kDa) in its proform and, depending on the treatment of the media, varying amounts of activated MMP-2 (65 kDa). This suggests that the increased gelatinolytic activity in keratoconus was not correlated with an increased appearance of either the 65-kDa-activated form of MMP-2 or a new MMP species. In addition, no differences in the amount of MMP-2 were detected that could account for the increased activities in keratoconus cultures. However, a relative decline in the detectable TIMP levels in keratoconus cultures resulted in an apparent three-fold increase in the ratio of MMP-2/TIMP. Northern blots showed no significant changes in mRNA levels for MMP-1, MMP-2, MMP-3, TIMP, or TIMP-2. These data suggest that a possible alteration in the interaction between MMP-2 and TIMP may play a role in the increased gelatinolytic activity seen in keratoconus tissues.

Identification of cell types in human diseased corneas.

PURPOSE: Activated myofibroblasts and macrophages are often found in corneal wound models. The current study was performed to determine whether human diseased corneas that had active tissue remodeling and enzyme activities also possessed myofibroblasts, macrophages, major histocompatibility complex class II cells, and/or CD-68-positive cells. METHODS: Normal, keratoconus, keratoconus with hydrops, bullous keratopathy, map-dot-fingerprint dystrophy, failed grafts, and acid burn/neovascularized corneas were collected, frozen in OCT, sectioned, and stained with antibodies to alpha smooth muscle actin (myofibroblast marker), CD14 (macrophage marker), CD68 (lysosomal membrane marker), and HLA-DR (major histocompatibility complex class II cells). Selective histochemical stains identified lysosomal enzymes. RESULTS: Normal and map-dot-fingerprint dystrophy corneas lacked antibody and enzyme staining. Keratoconus corneas were positive for CD68, HLA-DR, and lysosomal enzymes but were negative for CD14 and smooth muscle actin. Bullous keratopathy corneas had CD68-, CD14-, and HLA-DR-positive cells, relatively normal enzyme levels, and were smooth muscle actin-negative. Failed graft corneas had significant numbers of CD68-, CD14-, and HLA-DR-positive cells and increased acid phosphatase, but these corneas were smooth muscle actin-negative. Ulcerated and vascularized corneas had positive staining with all antibodies that were examined. Cultured stromal cells from normal corneas were CD68-positive, CD14-negative, and alpha smooth muscle actin-negative, and they produced lysosomal enzymes. CONCLUSIONS: The current study demonstrates that increased presence of lysosomal enzymes, corneal remodeling, and fibrosis can occur in the absence of myofibroblasts and/or macrophages.

Keratoconus corneas: increased gelatinolytic activity appears after modification of inhibitors.

We examined the metalloproteinase activity from normal and keratoconus corneal extracts. No differences were detected in the total amount of the metalloproteinase or its physical form of activation. However, there was a significant elevation in enzymatic activity in the keratoconus extracts after chemical modification of inhibitory elements. This suggests either a difference in the enzymatic capabilities of keratoconus corneas or, as suggested previously, a decrease in the amount of TIMP (tissue inhibitor of metalloproteinase) present in the tissue.

Localization of TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B in pathological human corneas.

PURPOSE: Determine the tissue distribution patterns for tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3), gelatinase A and gelatinase B in normal and pathologic corneas. METHODS: Corneas were examined by immunohistochemistry, using antibodies to TIMP-1, TIMP-2, TIMP-3, gelatinase A or gelatinase B. RESULTS: In normal corneas, TIMP-1 antibody stained the epithelium and endothelium. TIMP-2 and TIMP-3 stained the epithelium, keratocytes and endothelium. Gelatinase A staining was weak and restricted to the epithelial cells. Radial keratotomy scars showed increased staining for TIMP-1 and TIMP-2 around the epithelial cell plug and along the incision. Bullous keratopathy corneas showed TIMP staining patterns similar to normal corneas and increased gelatinase A staining in regions of subepithelial fibrosis. Stromal scars of keratoconus corneas also had increased staining with TIMP-1 and TIMP-2 antibodies. In many keratoconus corneas, the TIMP-3 staining pattern was similar to normal corneas. However, in some keratoconus corneas, when Bowman's layer was missing, the stroma beneath was completely devoid of TIMP-3 antibody staining. No gelatinase B was seen in either the normal or diseased corneas. CONCLUSION: These data suggest that TIMP-1 and TIMP-2 are important for scar formation and corneal remodeling, since they were found in increased amounts at radial keratotomy incision sites and keratoconus scars. The significance of the focal stromal defects in TIMP-3 staining, associated with absence of Bowman's layer on keratoconus corneas, needs to be elucidated. At the stages of disease examined in this study, gelatinase B may not play a significant role in these pathological processes, since it was not seen in any of the corneas examined.

Altered gelatinolytic activity by keratoconus corneal cells.

Keratoconus involves thinning and central protuberance of the cornea, scarring and significantly decreased vision. It is one of the major causes of corneal transplantation in this country, but the etiology of this disorder is unclear. In the present study stromal keratocytes were isolated and cultured from normal and keratoconus human corneas. Consistent with the phenotype of cornea thinning, we observed an increased gelatinolytic activity in keratoconus cultures. Characterization of enzyme properties in these cells suggested that gelatinase (type IV collagenase) was responsible for the majority of proteolytic activity found in this system. This elevated gelatinolytic activity was present in spite of lower amounts of total protein being produced by the keratoconus cultures.

Characterization of the major matrix degrading metalloproteinase of human corneal stroma. Evidence for an enzyme/inhibitor complex.

The human cornea is an avascular, highly organized tissue with the unique property of transparency. While the extracellular matrices of this tissue are composed of a variety of collagen types, proteoglycans and glycoproteins, little is known of the normal degradation and remodeling of these components. We examined the capacity of organ cultured human ocular tissues to produce and secrete metalloproteinases, a family of related enzymes capable of digesting a variety of extracellular matrices. We demonstrated that while enzymatic activities similar to type I collagenase and stromelysin are produced, the predominant activities of the corneal stroma and keratocyte cultures are a 68-kDa gelatinase. In our hands, this enzyme does not appear to be induced significantly by phorbol esters in vitro. In addition, this enzyme appears to be secreted as a complex with a 21-kDa protein that functions as an enzymatic inhibitor. Moreover, the keratocytes also produce a 28-kDa inhibitor which has similar properties to tissue inhibitor of metalloproteinase (TIMP).

Altered type VI collagen synthesis by keratoconus keratocytes in vitro.

Type VI collagen and gelatinase A are synthesized by human keratocytes in vitro. Keratoconus is a corneal disease that has increased gelatinase A activity. Normal keratocyte culture media analyzed by Western blotting with polyclonal antibody to type VI collagen showed a single 140 kDa band that increased in intensity after phorbol ester treatment. The same 140 kDa band was absent in media collected from keratoconus keratocytes cultured with or without phorbol ester. We speculate that in keratoconus keratocyte cultures, type VI collagen may be degraded or modified by the increased gelatinase A activity so as not to be recognized by the type VI antibody.

Enhanced expression of a transmembrane phosphotyrosine phosphatase (LAR) in keratoconus cultures and corneas.

The purpose of the study was to identify genes that are differentially expressed in normal versus keratoconus corneas. Total RNA isolated from corneal stromal cell cultures was reverse-transcribed and then amplified by the polymerase chain reaction (PCR) using defined, arbitrary primers. The products were displayed on polyacrylamide gels and bands that were differentially expressed were excised, re-amplified and subcloned. The resulting clones were sequenced and utilized as probes for Northern blots with cultured cell RNA or Southern blots of corneal cDNA. One of the products that appeared to be more highly expressed in keratoconus cultures and corneas displayed 100% homology with leukocyte common antigen related protein (LAR), a transmembrane phosphotyrosine phosphatase. Western analyses and immunohistochemistry with monoclonal and/or polyclonal antibodies to LAR were used to examine keratocyte cultures and fresh frozen normal, keratoconus and pseudophakic bullous corneas. We identified a gene product with 100% homology to LAR that is expressed at the RNA level in keratoconus corneas and cell cultures but is found only at low or undetectable levels in normal cultures and normal and pseudophakic bullous keratopathy (PBK) corneas. By Western blotting and immunofluorescence with specific LAR antibodies, the protein was identified in keratoconus stromal cell cultures but not in normal cultures. When fresh frozen tissue was examined, LAR protein was localized to numerous stromal cells throughout central keratoconus corneas, while no central staining was seen in normal or bullous keratopathy corneas. LAR, a transmembrane phosphotyrosine phosphatase, is more highly expressed in keratoconus corneas and stromal cell cultures as demonstrated by differential display, Northern analyses, immunohistochemistry and Western blotting.

Altered expression of growth factors and cytokines in keratoconus, bullous keratopathy and diabetic human corneas.

The purpose of this study was to identify the growth factors and cytokines present in normal and diseased corneas. Total RNA was isolated from normal and diseased corneas. cDNA was synthesized from individual corneas and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with primers to IL-1alpha, 1IL-8, PDGF-B, BMP-2, BMP-4, IGF-I, TGF-beta2, FGF-2, and VEGF. After normalization to beta2-microglobulin, several factors were identified that were significantly different from normal. Antibodies to IGF-I, BMP-2, VEGF and TGF-beta2 were used for immunohistochemistry. A total of 93 corneas were used for this study including 31 normal, 20 keratoconus, 19 bullous keratopathy (pseudophakic and aphakic, PBK/ABK), and 23 diabetic corneas. The VEGF RNA levels were significantly decreased in the keratoconus and PBK/ABK corneas but increased in the diabetic corneas. BMP-2 gene expression was lower than normal in the PBK/ABK and diabetic corneas. IGF-I and BMP-4 RNA levels were increased in PBK/ABK. In the immunohistochemical studies, the protein patterns paralleled those found at the mRNA level. The only exception was IGF-I in diabetic corneas that showed increased staining in the epithelium and its basement membrane without a significant increase in mRNA levels. TGF-beta2 mRNA and protein levels were similar to normal in all diseased corneas. Thus, no alterations in the tested growth factors/cytokines were unique to keratoconus corneas. In contrast, PBK/ABK corneas had specific significant elevations of BMP-4 and IGF-I. Diabetic corneas were unique in their increased VEGF mRNA levels. These data suggest that while some growth factor/cytokine alterations are non-specific and can be found in multiple corneal diseases, there are others that are unique to that disease.

Overexpression of matrix metalloproteinase-10 and matrix metalloproteinase-3 in human diabetic corneas: a possible mechanism of basement membrane and integrin alterations.

We have previously described decreased immunostaining of nidogen-1/entactin; laminin chains alpha1, alpha5, beta1,gamma1; and epithelial integrin alpha3beta1 in human diabetic retinopathy (DR) corneas. Here, using 142 human corneas, we tested whether these alterations might be caused by decreased gene expression levels or increased degradation. By semiquantitative reverse transcription-polymerase chain reaction, gene expression levels of the alpha1, alpha5, and beta1 laminin chains; nidogen-1/entactin; integrin alpha3 and beta1 chains in diabetic and DR corneal epithelium were similar to normal. Thus, the observed basement membrane and integrin changes were unlikely to occur because of a decreased synthesis. mRNA levels of matrix metalloproteinase-10 (MMP-10/stromelysin-2) were significantly elevated in DR corneal epithelium and stroma, and of MMP-3/stromelysin-1, in DR corneal stroma. No such elevation was seen in keratoconus corneas. These data were confirmed by immunostaining, zymography, and Western blotting. mRNA levels of five other proteinases and of three tissue inhibitors of MMPs were similar to normal in diabetic and DR corneal epithelium and stroma. The data suggest that alterations of laminins, nidogen-1/entactin, and epithelial integrin in DR corneas may occur because of an increased proteolytic degradation. MMP-10 overexpressed in the diabetic corneal epithelium seems to be the major contributor to the observed changes in DR corneas. Such alterations may bring about epithelial adhesive abnormalities clinically seen in diabetic corneas.