Immunolocalization of type VI collagen in developing and healing rabbit cornea.

We have localized type VI collagen in normal developing and corneal scar tissue. Indirect immunofluorescence showed that type VI collagen was distributed throughout the normal stroma and most of the scar. No fluorescence was detected along the posterior margin of the scar and in a retrocorneal membrane continuous with the scar. Since the corneal endothelium in rabbits contributes to the formation of scar tissue and retrocorneal membrane, our observations suggest that the endothelium does not synthesize type VI collagen. Indirect immunoelectron microscopy showed that type VI collagen was located abundantly between collagen fibrils as fine filamentous structures containing beads with a periodicity of 100 nm, consistent with published observations of other tissues. Because these filaments are more prominent when stained with ruthenium red, and predigestion of tissue with Chondroitinase ABC enhances binding of monoclonal antibody to type VI collagen, proteoglycans probably are associated with this collagen in the cornea. Ultrastructural observations supported by previous biochemical analyses show that the proportion of type VI collagen to fibrillar collagen is smaller in scar tissue compared with fetal cornea. The abundance of type VI collagen and its distribution and association with proteoglycans in rabbit corneal tissues suggest that this macromolecule plays a role in the tensile strength and transparency of the stroma.

Alpha-1 proteinase inhibitor levels in keratoconus.

The levels of alpha 1-proteinase inhibitor (alpha 1-antitrypsin) in keratoconus, normal human, and other diseased corneas were examined. Using an immunoperoxidase technique, the presence of this inhibitor was demonstrated in the epithelium, stroma and endothelium of all corneal sections. Compared with normal human controls, the staining intensity in the epithelium and stromal lamellae of keratoconus corneas was markedly reduced. Such a reduction was not seen in either scarred or other diseased corneas. Extracts of keratoconus and normal human corneas were subsequently analyzed for alpha 1-proteinase inhibitor by a dot blot assay using a monoclonal antibody against the inhibitor and a 125I-labelled secondary antibody. In agreement with the immunohistochemical findings, the alpha 1-proteinase inhibitor level found in the epithelium of keratoconus corneas was approximately one-fourth of that found in normal human controls. In addition, the stromal extracts of keratoconus corneas contained about one-sixth the inhibitor level of that in normal human extracts. These results lend further support to the hypothesis that degradation processes may be aberrant in keratoconus.

Corneal stromal wound healing in rabbits after 193-nm excimer laser surface ablation.

An argon fluoride excimer laser (193 nm) with a moving slit delivery system was used to perform anterior myopic keratomileusis in both eyes of 24 New Zealand white rabbits. Rabbits were killed immediately after ablation and at intervals up to 100 days. By slit-lamp microscopy, four rabbits at day 100 exhibited four clear corneas and four corneas had central, spotty, subepithelial haze. Light and electron microscopy documented corneal healing. In the early stages a transient acellular zone in the anterior stroma appeared over a period of three weeks, followed by an increased number of fibrocytes. In the corneas with opacification, focal areas of 20-microns-thick subepithelial scarring were present. An unexpected finding was transient damage to posterior stromal keratocytes and endothelial cells. The endothelium produced a layer of granular material that migrated anteriorly across Descemet's membrane. Immunochemistry at day 6 showed a marked staining for collagen IV, proteoglycans, fibronectin, and laminin.

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.

Modulation of HLA antigen expression on corneal epithelial and stromal cells.

Human corneal epithelial cells and stromal fibroblasts in culture were incubated with gamma interferon or with medium conditioned by phytohemagglutinin (PHA)-stimulated mononuclear cells. The corneal cells were placed into suspension, assayed for class I (HLA-A,B,C) and class II (HLA-DR) antigens by indirect immunofluorescence, and analyzed with flow cytometry. Epithelial cells treated for 5 days with conditioned medium (CND-M) did not exhibit an increase in class I or an induction of class II antigen expression, although a trend toward increased class I antigen expression was present. Epithelial cells treated for 5 days with 250-500 U/ml of gamma interferon did not demonstrate an increase in class I but did show an induction of class II antigen expression; again, however, a trend toward increased class I antigen expression was present. Stromal fibroblasts treated for 3-5 days with CND-M exhibited an increase in class I antigen expression, but stromal fibroblasts treated for 1-5 days with CND-M did not show an induction of class II antigen expression. Stromal fibroblasts incubated for 1-5 days with 250-750 U/ml of gamma interferon demonstrated both an increase in class I and an induction of class II antigen expression. These data suggest that host lymphokines may intensify the process of corneal graft rejection by augmenting class I antigen expression on allogeneic cells. Moreover, the induction of class II antigen expression by host lymphokines on cells in transplanted corneal tissue may lead to host sensitization and subsequent allograft rejection.

Bovine corneal stroma contains a structural glycoprotein located in the gap region of the collagen fibrils.

Treatment of bovine corneal stroma using SDS-containing extracting solutions removes a 135,000 MW glycoprotein from the main collagen framework of the tissue. Low-angle synchrotron X-ray diffraction patterns obtained from corneas extracted in this way indicate that the glycoprotein has been removed from the gap regions of the collagen fibrils and is thus an important structural component of the corneal stroma. The glycoprotein (GP 135) shares a number of properties with one of the subunits of type VI collagen, but tests have so far failed to establish their identity.

A large-scale, orthogonal network of microfibril bundles in the corneal stroma.

Orthogonal and parallel arrays of microfibril bundles are described in the corneal stroma of embryonic and adult chickens. The arrays lie parallel to the corneal surface and are distributed in the extracellular matrix between Bowman's layer and Descemet's membrane. The individual microfibril bundles measure 0.1-0.25 micron in diameter and consist of 20-30 "tubular" rods, of approximately 15 nm diameter, and an apparently structureless matrix. The arrays have a spacing of approximately 3 microns. We speculate that the microfibril bundles serve as a scaffolding for the corneal stroma or as a light-diffracting element. With some variations in distribution and organization, the arrays of microfibril bundles also occur in calf, rabbit, rat, newborn mice, toad, and goldfish, but not in adult human corneas.

Distribution of fibronectin in human and rabbit corneas.

In order to study the possible role of fibronectin (FN) in corneal wound healing and the relationship between FN and sensory innervation, FN was demonstrated immunohistochemically in both normal and sensorily denervated rabbit corneas and in normal or tissue-cultured human corneas. The distribution of FN was the same in the groups examined: a thin subepithelial band of FN-like immunoreactivity was seen at the level of epithelial basement membrane and at the stromal side of Descemet's membrane. Epithelial abrasions were also performed in both normal and denervated rabbit corneas. The results were compared with those obtained from organ-cultured human corneas. Following abrasion of the corneal epithelium, FN was detected in the anterior margin of the denuded stroma 18 hr after the operation in the areas where the epithelium had not healed, but not 49 hr after. Sensory denervation did not affect the distribution of FN in normal, denervated or healing rabbit cornea. It is concluded that FN is probably not controlled by sensory innervation.

Immunolocalization of keratan sulphate in the human embryonic cornea and other human foetal organs.

An antibody raised against keratan sulphate was used to localize keratan sulphate in the human embryonic cornea and other human foetal organs. It was found that keratan sulphate was present in the corneal endothelium, as well as in the corneal stroma. In contrast, no keratan sulphate could be demonstrated in the corneal epithelium or in any posterior parts of the human foetal eye. Human corneal stromal cells grown in monolayers in tissue culture lost their capacity to bind this antibody. This suggests that explantation in vitro decreases or alters the expression of keratan sulphate in this cell type. Keratan sulphate was found to be present in abundant quantities in human embryonic cartilage and to a lesser extent in placenta. In contrast, keratan sulphate could not be detected in the foetal intestine, liver, suprarenal glands, the umbilical cord or in the foetal yolk sack.

Immunolocalization of type IV collagen and laminin in nonbasement membrane structures of murine corneal stroma. A light and electron microscopic study.

The extracellular matrix of adult vertebrate corneal stroma is composed primarily of the interstitial collagen type I and smaller amounts of types III and V collagen. These collagens are organized into overlapping lamellae of striated filaments. In addition to these lamellar structures, the corneal stroma also contains 100- to 250-nm bundles of nonstriated 8- to 11-nm microfibrils. By immunofluorescent localization and electron microscopic immunolocalization, these microfibril bundles in the mouse are associated with type III collagen, type IV collagen, and laminin. By immunologic and histochemical criteria, these bundles do not contain either type I collagen, type V collagen, elastin, or oxytalan microfibrils. The cellular source, composition, and possible functions of these microfibril bundles are discussed.

Heterogeneity, polydispersity, and physiologic role of corneal proteoglycans.

Gel chromatography, affinity chromatography, ultracentrifugation, enzymic fragmentation, and analysis of amino acids, hexosamines and neutral sugars were used to characterize a heterogeneous fraction of proteoglycans from bovine corneal stroma. The results indicate that the fraction largely consists of a mixture of the 2 main types of corneal proteoglycans described earlier, namely keratan sulfate proteoglycans and chondroitin sulfate-rich proteoglycans with covalently bound oligosaccharides. Models for the structure of proteoglycans are suggested, an it is concluded that the molecular size of corneal proteoglycans makes them appropriate as 'spacers' between the collagen fibrils, a property important for corneal transparency. Cornea is softer than cartilage because corneal proteoglycans are less underhydrated than cartilage proteoglycans.