cathepsin-g and Keratoconus

Keratoconus is a debilitating corneal thinning disease that principally develops in the second and third decades of life. Our group previously developed a novel approach to studying keratoconus, based on the observation that there is a gradient of damage across the keratoconic cone. We identified a number of cellular characteristics of keratoconus such as discrete incursions of fine cellular processes from the anterior keratocytes in association with localised indentation of the basal epithelium, and increased levels of the lysosomal enzymes Cathepsin B and G in aberrant keratocytes, located beneath compromised regions of Bowman's layer, but also deeper in the stroma. Enzyme activity by these cells seemed to be causing localised structural degradation of the anterior stroma, leading to near-complete destruction of both Bowman's layer and the stroma, often necessitating a full-thickness corneal graft for sight restoration. This current study extends our initial findings by investigating the role of corneal nerves passing between the stroma and epithelium at the sites of early degradative change observed previously, and may be facilitating the keratocyte-epithelial interactions in this disease. Cells in sections of normal and keratoconic human corneas were labelled with the fixable fluorescent viability dye 5-chloromethylfluorescein diacetate, antibodies to alpha-tubulin (nerves), alpha3beta1 integrin, Cathepsin B and G, and the nuclear dye DAPI, and then examined with a confocal microscope. Anterior keratocyte nuclei were seen wrapping around the nerves as they passed through the otherwise acellular Bowman's layer, and as the disease progressed and Bowman's layer degraded, these keratocytes were seen to express higher levels of Cathepsin B and G, and become displaced anteriorly into to the epithelium. Localised nerve thickenings also developed within the epithelium in association with Cathepsin B and G expression, and appeared to be very destructive to the cornea. Insight into the molecular mechanisms of keratoconic disease pathogenesis and progression can be gained from the process of extracellular matrix remodelling known from studies of connective tissues other than the cornea, and wound healing studies in the cornea. Further studies are required to determine how well this model fits the actual molecular basis of the pathogenesis of keratoconus.

Topics: Cathepsin B; Cathepsin G; Cathepsins; Cornea; Disease Progression; Epithelium; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Keratoconus; Microscopy, Confocal; Serine Endopeptidases

Analysis of corneal tissue from normal and keratoconic donors has revealed differences which may represent early signs in the pathogenesis of keratoconus. Peripheral areas of keratoconic tissue obtained from transplant surgery were targeted to ascertain cellular disposition and morphological changes which may be masked within the extensive damage of the central keratoconic cone. Peripheral keratoconic corneae exhibited discrete incursion of fine cellular processes into Bowman's membrane. These processes originated from keratocytes and were often observed in conjunction with a defined indentation from the basal epithelium. Comparison of the lysosomal enzymes cathepsin B and G with constitutively expressed cytoplasmic esterase determined that both cathepsins were elevated within keratocytes of keratoconic tissue compared with normal tissue. Some clusters of keratoconic keratocytes had elevated levels of cathepsin exceeding all others. Cathepsin-rich keratocytes localized with morphologically compromised regions of Bowman's membrane. The presence of cell nests deeper within the stroma indicated that the catabolic changes, which are visible within the acellular Bowman's membrane, are probably also occurring deeper within the stroma, but are masked and not readily detectable.

Topics: Cathepsin B; Cathepsin G; Cathepsins; Cornea; Epithelium, Corneal; Humans; Image Processing, Computer-Assisted; Keratoconus; Microscopy, Confocal; Serine Endopeptidases

Keratoconus is characterized by thinning and scarring of the central region of the cornea. The authors have shown, in corneas obtained from patients with keratoconus, that lysosomal enzyme activities are elevated, whereas levels of protease inhibitors such as alpha 1-proteinase inhibitor (alpha 1-PI) are reduced. This study was undertaken to examine further the gene expression of cathepsin G, acid phosphatase, and alpha 1-PI in keratoconus corneas.. Corneal buttons were collected from patients with keratoconus, normal subjects, and patients with other corneal diseases. In situ hybridization was performed on paraffin sections using a tritium-labeled probe for cathepsin G or alpha 1-PI. Competitive polymerase chain reaction (PCR) was used to determine the messenger RNA (mRNA) levels for lysosomal acid phosphatase and alpha 1-PI in epithelial and stromal cells of keratoconus corneas.. Silver grains, indicative of positive in situ hybridization products, were observed in all three cell types of normal corneas for both DNA probes. Compared with normal and other diseased controls, the labeling was enhanced for cathepsin G but was diminished for alpha 1-PI in the epithelium of keratoconus corneas. Competitive PCR showed that the mRNA level for acid phosphatase was higher and that the mRNA level for alpha 1-PI was lower in keratoconus corneas.. These results indicate that the mRNA level for degradative enzymes in increased and that for alpha 1-PI it is reduced in keratoconus corneas. This study provides the first evidence that the altered expression of multiple enzymes and inhibitors in keratoconus occurs at the gene level. Furthermore, it implicates a possible role of coordinated transcriptional regulation of gene expressions in keratoconus.

Topics: Acid Phosphatase; Adolescent; Adult; Aged; Aged, 80 and over; alpha 1-Antitrypsin; Cathepsin G; Cathepsins; Child; Child, Preschool; Cornea; DNA Primers; DNA Probes; Electrophoresis, Agar Gel; Gene Expression Regulation, Enzymologic; Humans; In Situ Hybridization; Keratoconus; Middle Aged; Polymerase Chain Reaction; RNA, Messenger; Serine Endopeptidases; Serine Proteinase Inhibitors