Charles Cintron

Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea.

We have documented changes in collagenolytic/gelatinolytic enzymes of the matrix metalloproteinase family (MMP) in remodelling rabbit cornea. MMP-2 (65 kDa gelatinase) in the proenzyme form is synthesized by the cells of the normal corneal stroma. After keratectomy the level of MMP-2 is increased in the stroma and enzyme appears in both pro- and activated forms. In addition, corneal cells synthesize MMP-9 (92 kDa gelatinase) in the proenzyme form after keratectomy; expression occurs in both the epithelial as well as stromal corneal layers. Changes in expression of both enzymes are precisely localized to the repairing portion of cornea, but demonstrate important differences in timing that correlate with the timing of specific events of matrix remodelling. Our data suggest that each of the gelatinases plays a different role in tissue remodelling after injury. We hypothesize that MMP-2 performs a surveillance function in normal cornea, catalyzing degradation of collagen molecules that occasionally become damaged. After wounding, this enzyme appears to participate in the prolonged process of collagen remodelling in the corneal stroma that eventually results in functional regeneration of the tissue. MMP-9 expression does not correlate with stromal remodelling, but we suggest that the enzyme might play a part in controlling resynthesis of the epithelial basement membrane.

βig-h3 is synthesized by corneal epithelium and perhaps endothelium in Fuchs' dystrophic corneas

PURPOSEnDeposition of abnormal sub-epithelial matrix and posterior collagenous layer by epithelium and endothelium, respectively, in Fuchs dystrophy gives us the opportunity to determine if these tissues synthesize beta ig-h3.nnnMETHODSnImmunohisto-/immunocytochemistry of corneas were conducted with rabbit anti-human beta ig-h3 and monoclonal anti-human type VI collagen. Labeled sense and anti-sense beta ig-h3 oligonucleotide probes were used for in situ hybridization.nnnRESULTSnbeta ig-h3-specific fluorescence was found just beneath detached epithelium in the sub-epithelial matrix, abnormal Descemets membrane and posterior collagenous layer. Type VI collagen co-localized with beta ig-h3 within abnormal sub-epithelial matrix and corneal stroma adjacent to Descemets membrane. beta ig-h3 mRNA was detected in corneal epithelium of dystrophic corneas.nnnCONCLUSIONSnExpression of beta ig-h3 in sub-epithelial matrix and posterior collagenous layer of Fuchs dystrophy is consistent with the synthesis of new extracellular matrices by epithelial and endothelial tissues. beta ig-h3 mRNA in corneal epithelium further supports an epithelial source of this protein. Endothelial synthesis of beta ig-h3 is based on circumstantial evidence due to cell loss during surgical and histological procedures. Co-localization of beta ig-h3 with type VI collagen in abnormal sub-epithelial matrix and at the stromal/Descemets membrane interface suggest this collagen in association with beta ig-h3 interacts with these tissues and anchors them to the adjacent stroma.

Quantitative studies of corneal epithelial wound healing in rabbits

Corneal epithelial defects are covered rapidly by the movement of adjacent epithelium. However, the mechanism of this tissue movement is poorly understood. In this study, the quantity or cell water, protein, and DNA were determined in healing epithelium to test the hypothesis that cell enlargement contributes to the rapid coverage of the defect. In addition, light and transmission electron microscopy and [3H] thymidine incorporation into epithelial cells were used to determine whether the healing tissue moves as a unit or as individual cells. The quantitative determinations lead us to conclude that healing begins with a dramatic rise in cell water, followed by an increase in cell protein and finally by a gradual increase in DNA. The morphologic and autoradiographic evidence strongly suggests that large corneal epithelial defects in rabbits are covered by the movement of adjacent tissue as a unified, multilayered sheet of cells. Furthermore, the cells appear large than normal with minimal changes in intercellular spaces. We suggest that the increase in cell volume is due to water uptake, which plays an important role in covering the defect by increasing the cells surface area. Protein is then accumulated, followed by cell proliferation.

Fibronectin in developing rabbit cornea

Fibronectin is believed to be important in tissue morphogenesis. We examined the distribution of fibronectin in developing rabbit cornea by immunohistofluorescence. Cryostat sections of cornea from 13, 15, and 20-day-old fetuses, 3-day neonates, and adults were incubated with affinity-purified fluoresceinated guinea pig anti-rabbit fibronectin antiserum (aFN). aFN bound to components within the presumptive stromal region and along the basal surfaces of corneal and lens epithelia during early stages of mesenchymal invasion. At 15 days of gestation, fluorescence was associated with the stromal extracellular matrix of the cornea, the subepithelial zone, and the lens capsule. In the 20-day fetus an intense aFN fluorescence was present along the inner corneal stromal border coincident with the formation of Descemets membrane. Fluorescence within the corneal stroma appeared as fine lines, restricted to the collagen lamellae, remaining through birth and disappearing in the adult. Although stromal fluorescence disappeared in the adult, Descemets membrane continued to fluoresce, albeit to a lesser extent. The results of our studies indicate the presence of fibronectin in developing rabbit cornea. Because fibronectin is important to cell adhesion in vitro, and because intercellular and cell-extracellular matrix interactions, including adhesion, are necessary for tissue morphogenesis, our observation suggests that fibronectin plays an important role in corneal morphogenesis.

Changes in chemical composition of anterior lens capsules of cataractous human eyes as a function of age

Abstract Amino acids and carbohydrates were found to account for 80% and 11%, respectively, of the dry weight of the anterior lens capsule of the cataractous human lens. The human lens capsule was found to contain substantial amounts of glycine, hydroxyproline, proline and glycosylated hydroxylysine, as is typical of basement membranes. The monosaccharides present in the lens capsule were identified as glucose, galactose, fucose, mannose, glucosamine, galactosamine and sialic acid. In contrast with basement membranes studied so far, hydroxylysine in human anterior lens capsules from cataractous lenses is almost completely glycosylated. Lenses used in this study were divided into two age groups: ≤ 65 years and &> 65 years. There were statistically significant differences between capsules from the two groups. In the older group, amounts of tyrosine, phenylalanine, histidine, lysine, and arginine were significantly higher and hydroxyproline was significantly lower. These compositional differences are consistent with a smaller proportion of collagen in the older age group.

Localization of type XII collagen in normal and healing rabbit cornea by in situ hybridization

To identify the cell types responsible for type XII collagen synthesis in normal and healing rabbit cornea, a partial cDNA sequence of rabbit type XII collagen, obtained from an adult rabbit cornea cDNA library, was used to develop highly specific oligonucleotide probes for Northern blot analysis and in situ hybridization. Approximately 2000 bases of a type XII collagen 2.2 kb cDNA clone were sequenced. Comparative sequence analysis of the bases showed a 74% identity with chick alpha 1 (XII) chain of type XII collagen. The deduced amino acid sequence indicated a 72% identity with chick type XII collagen. Northern blot analysis showed that cultures of cornea stromal and endothelial cells each contain two RNA species, greater than 10 kb, that hybridize to rabbit type XII collagen oligonucleotide probes. Although normal stromal cells failed to show type XII collagen mRNA, normal endothelial cells contain mRNA for this collagen. These results indicate that endothelium of normal rabbit cornea has a potential to synthesize type XII collagen. During corneal wound healing, both endothelium-derived and stroma-derived cells in the developing scar tissue contained type XII mRNA. In view of the known presence of type XII collagen in corneal stromas from chick and mouse, the distribution of mRNA in normal cornea is puzzling.

Immunohistochemistry of Proteoglycans in Human Lamina Cribrosa

Proteoglycans are macromolecular components of connective tissue, which are believed to have an important role in the organization of other extracellular matrix components and in the hydration and rigidity of tissue. Using antibodies to the heparan sulfate-, dermatan sulfate-, and keratan sulfate-proteoglycans core proteins, we used indirect immunofluorescence to determine the presence of these core proteins and to characterized their distribution in the lamina cribrosa of healthy human eyes of various ages. Findings with heparan sulfate-proteoglycan core proteins support the multilayered organization of basement membranes in the lamina cribrosa as proposed in previous reports. Dermatan sulfate-proteoglycan core protein results indicate colocalization with fibrillar collagen. Application of monoclonal antibodies to the glycosaminoglycan side chain disclosed an increasing predominance of chondroitin-4-sulfate over chondroitin-6-sulfate with age. This may indicate a decreasing water-holding capacity of the cribriform plates and suggest a decreasing ability to withstand pressure. This study attempts to identify the core proteins of proteoglycans in human lamina cribrosa and serves as a basis for study of glaucomatous eyes.

Disparate Effects of Calmodulin Inhibitors on Corneal Epithelial Migration in Rabbit and Rat

We investigated the effects of two calmodulin inhibitors, trifluoperazine and N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide, on corneal epithelial wound closure in rabbits and rats. Measured, round epithelial defects were made on corneas by gentle scraping. After 20 h in organ culture with or without inhibitor, the remaining wound area was measured and samples were fixed for microscopy. In the rabbit, these inhibitors had little or no effect on the rate of wound coverage. In the rat, however, both trifluoperazine (3-40 microM) and N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (10-100 microM) inhibited wound closure in a dose-dependent manner. Thus we believe that calmodulin plays a crucial role in epithelial migration in the rat. In the rabbit, there seems to exist a mechanism that is not critically dependent upon an intact calmodulin pathway.

Microcrystalline Collagen and Corneal Wound Healing

Inserting 0.45 mg of microcrystalline collagen (MCC) into a freshly trephined rabbit corneal wound immediately seals the lesion with no furthur loss of aqueous humor. Analysis of MCC and control scars 2 and 3 weeks after wounding showed no significant differences in dry weight, proline and hydroxyproline content or incorporation of 3H-proline into collagen and noncollagen proteins. After 2 weeks DNA increased in MCC-treated wounds, corresponding to an increased number of cells. Some of these cells appear to remove MCC while the healing process proceeds.