acid-phosphatase and Keratoconus

Keratoconus is a progressive disease that thins and scars the corneal stroma. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of the inhibitors alpha1-proteinase inhibitor (alpha 1-PI) and alpha 2-macroglobulin (alpha 2-M) are reduced, especially in the epithelial layer. An increased expression of the transcription factor Sp1 was also demonstrated. The role of Sp1 in regulation of the genes affected in keratoconus was examined in this study.. DNA segments, containing 5'-flanking promoter sequences of the alpha 1-PI, LAP, cathepsin B, and alpha 2-M genes were ligated into the secreted alkaline phosphatase (SEAP) reporter gene vector. These constructs, along with the pSV beta-galactosidase control vector, were transfected into cultured human corneal epithelial and stromal cells and skin fibroblasts. Cotransfection with the Sp1 expression vector was performed in parallel. SEAP and beta-galactosidase enzyme activities were assayed.. In corneal epithelial cells, as in stromal cells, alpha 1-PI promoter activity was suppressed by cotransfection of pPacSp1. The LAP, cathepsin B, and alpha 2-M promoters were functional in corneal cells, whereas activities of these promoters were much lower in skin fibroblasts. Cotransfection experiments indicated that the up- or downregulation of LAP, cathepsin B, and alpha 2-M observed in keratoconus-affected corneas was not mediated by Sp1.. These results support the theory that the corneal epithelium, along with the stroma, is involved in keratoconus. An upstream role of Sp1 is indicated and the Sp1-mediated downregulation of the alpha 1-PI gene may be a key event in the disease development.

Topics: Acid Phosphatase; Adult; Alkaline Phosphatase; alpha 1-Antitrypsin; alpha-Macroglobulins; beta-Galactosidase; Cathepsin B; Child; Corneal Stroma; DNA Primers; Epithelium, Corneal; Fibroblasts; Gene Expression Regulation; Genetic Vectors; Humans; Keratoconus; Promoter Regions, Genetic; Skin; Sp1 Transcription Factor; Transfection

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

Keratoconus is a disease characterized by thinning and scarring of the central portion of the cornea. We have shown, in corneas and conjunctival tissues obtained from patients with keratoconus, that lysosomal enzyme staining is enhanced in the epithelial layer. In this study, we examined the lysosomal enzyme staining in skin-biopsy specimens of patients with keratoconus. Tissues collected from four patients with keratoconus and six normal subjects were fixed and processed for histochemical staining. Results showed that the epidermal and dermal layers of all skin specimens stained positively for two lysosomal hydrolases, acid esterase and acid phosphatase. The enzyme staining in the skin epidermal and dermal cells of the keratoconus patients was within the normal range. The staining in the corneal and conjunctival epithelia of the same patients was more prominent than that of normal controls. It appears that the biochemical abnormality identified in keratoconus is not manifested in the skin.

Topics: Acetylesterase; Acid Phosphatase; Adult; Female; Histocytochemistry; Humans; Keratoconus; Lysosomes; Male; Middle Aged; Skin; Staining and Labeling

To examine the lysosomal enzyme activities in the conjunctival tissues of patients with keratoconus.. Tissues collected from 11 patients with keratoconus, eight patients with senile cataract, three patients with Fuchs' corneal dystrophy, and 11 normal control subjects were processed for histochemical staining for two lysosomal hydrolases, acid esterase and acid phosphatase.. The epithelium of all conjunctival specimens stained positively for the two enzymes. The staining in the conjunctival tissues of patients with keratoconus was more prominent than that seen in specimens from either normal control subjects or patients with other diseases.. Our results suggest that the conjunctival epithelium may be altered in keratoconus. Elevation of lysosomal enzyme levels has been demonstrated in the epithelium of corneas with keratoconus, implicating a role of this layer in the disease. The conjunctival abnormality seems to corroborate the corneal epithelial theory. It also adds one dimension to the pathogenesis of keratoconus.

Topics: Acetylesterase; Acid Phosphatase; Adult; Aged; Aged, 80 and over; Cataract; Conjunctiva; Epithelium; Female; Fuchs' Endothelial Dystrophy; Histocytochemistry; Humans; Keratoconus; Male; Middle Aged

We studied the lysosomal enzyme activities in corneas obtained from 12 patients with keratoconus. Three acid hydrolases--acid phosphatase, acid esterase, and acid lipase--were demonstrated by histochemical staining methods in the epithelium, stroma, and endothelium of corneas with keratoconus and normal human corneas. Analyses by an image-processing system indicated that the epithelium, especially the basal epithelium, of corneas with keratoconus had significantly higher levels of acid phosphatase, acid esterase, and acid lipase than those in normal human controls. Such an abnormality was not seen in either scarred corneas or corneas obtained from patients with Fuchs' corneal dystrophy. Our finding is consistent with the hypothesis that tissue degradation processes may be abnormal in keratoconus. In addition, our data suggest that the corneal epithelium, as previously speculated, may also be involved in this corneal disease.

Topics: Acetylesterase; Acid Phosphatase; Adolescent; Adult; Aged; Epithelium; Female; Histocytochemistry; Humans; Keratoconus; Lipase; Lysosomes; Male; Middle Aged