keratan-sulfate and Keratoconus

The corneal stroma is primarily composed of collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs). It is known that corneal crosslinking (CXL) treatment improves mechanical properties of the cornea. However, the influence of stromal composition on the strengthening effect of CXL procedure has not been thoroughly investigated. The primary objective of the present research was to characterize the effect of keratan sulfate (KS) GAGs on the efficacy of CXL therapy. To this end, the CXL method was used to crosslink porcine corneal samples from which KS GAGs were enzymatically removed by keratanase II enzyme. Alcian blue staining was done to confirm the successful digestion of GAGs and uniaxial tensile experiments were performed for characterizing corneal mechanical properties. The influence of GAG removal and CXL treatment on resistance of corneal samples against enzymatic pepsin degradation was also quantified. It was found that removal of KS GAGs significantly softened corneal tensile properties (P < 0.05). Moreover, the CXL therapy significantly increased the tensile stiffness of GAG-depleted strips (P < 0.05). GAG-depleted corneal buttons were dissolved in the pepsin digestion solution significantly faster than control samples (P < 0.05). The CXL treatment significantly increased the time needed for complete pepsin digestion of GAG-depleted disks (P < 0.05). Based on these observations, we concluded that KS GAGs play a significant role in defining tensile properties and structural integrity of porcine cornea. Furthermore, the stiffening influence of the CXL treatment does not significantly depend on the density of corneal KS GAGs. The findings of the present study provided new information on the relation between corneal composition and CXL procedure mechanical effects.

Topics: Animals; Collagen; Cornea; Corneal Stroma; Cross-Linking Reagents; Glycosaminoglycans; Keratan Sulfate; Keratoconus; Pepsin A; Photosensitizing Agents; Riboflavin; Swine; Ultraviolet Rays

Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) present in the corneal stroma where it is thought to regulate collagen fibril diameter. In this study we investigated the distribution of KS in normal and keratoconic corneas.. Four normal, one mild, and four severe keratoconic corneas were used for the study. Distribution of keratan sulphate proteoglycans (KS-PG) was investigated using a primary monoclonal antibody (5-D-4) that recognizes disulphated disaccharides in the poly-N-acetyllactosamine repeats of KS. The immuno-reactivity of 5-D-4 was analyzed by immunohistochemistry and immuno-electron microscopy.. Immuno-histochemistry showed diffuse 5-D-4 staining in keratoconic cornea compared to the punctuate staining in normal corneas. In the single cornea with mild keratoconus, immunogold microscopy revealed a very high density of KS-PG staining, especially in the posterior stroma, compared to severe keratoconic and normal cornea. The amount of KS-PG in the stroma in severe keratoconus was slightly less compared to the normal cornea. In the mild keratoconic cornea, a higher quantity of KS-PG was present around the keratocytes. In severe keratoconic corneas, a higher quantity of KS-PG was present within the keratocytes compared to normal cornea.. The finding of an altered expression of KS in our keratoconic corneas, in particular the strong expression of KS in keratocytes, is in keeping with reports of an altered expression of proteoglycan metabolism in keratoconus. KS-PG plays an important role in stromal collagen fibril assembly and a dysregulation of KS-PG synthesis or catabolism could explain changes in collagen fibril spacing and diameter, which we have reported elsewhere.

Topics: Adult; Aged; Antibodies, Monoclonal; Bowman Membrane; Cell Membrane; Cornea; Corneal Stroma; Descemet Membrane; Epithelium, Corneal; Fluorescent Antibody Technique, Indirect; Humans; Keratan Sulfate; Keratoconus; Microscopy, Fluorescence; Microscopy, Immunoelectron; Middle Aged; Polysaccharides; Sulfates; Young Adult

Macular corneal dystrophy (MCD) is an autosomal recessive inherited disorder that is accompanied by corneal opacity. Explants from MCD-affected corneas have been reported to synthesize low-sulfated KS, suggesting that sulfate groups attached to KS may play critical roles in maintaining corneal transparency. To clear the biosynthetic defect in the MCD cornea, sulfotransferase activities were determined that are presumably involved in the biosynthesis of KS: galactose-6-sulfotransferase (Gal6ST) activity and N-acetylglucosamine 6-O-sulfotransferase (GlcNAc6ST) activity.. Gal6ST and GlcNAc6ST activities, which were contained in the corneal extracts from corneas affected by MCD and keratoconus and from normal control corneas, were determined by measuring the transfer of (35)SO(4) from [(35)S]3'-phosphoadenosine 5'-phosphosulfate into the Gal residue of partially desulfated KS and the nonreducing terminal GlcNAc residue of GlcNAcbeta1-3Galbeta1-4GlcNAc (oligo A), respectively.. The level of Gal6ST activity in corneal extracts from eyes with MCD, which was measured by using partially desulfated KS as an acceptor, was nearly equal to that in eyes with keratoconus and normal control eyes. In contrast, GlcNAc6ST activity in the extracts from MCD-affected corneas, which was measured by using oligo A as an acceptor, was much lower than in those in corneas with keratoconus and in normal control corneas.. The decrease in GlcNAc6ST activity in the cornea with MCD may result in the occurrence of low- or nonsulfated KS and thereby cause corneal opacity.

Topics: Adult; Aged; Carbohydrate Sulfotransferases; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Paper; Cornea; Corneal Dystrophies, Hereditary; Female; Fluorescent Antibody Technique, Indirect; Humans; Keratan Sulfate; Keratoconus; Keratoplasty, Penetrating; Male; Middle Aged; Sulfotransferases

Five consecutive patients underwent epikeratoplasty for keratoconus. Postoperatively, four patients had poor visual acuity (average, 20/200) secondary to folds in Descemet's membrane and interface scarring. Two underwent penetrating keratoplasty eight months later. Histopathologic examination of the host corneas and the overlying lenticules disclosed epithelial irregularity and subepithelial fibrosis. The host corneas showed folds in Descemet's membrane and focal posterior stromal fibrosis. Electron microscopy disclosed breaks in Bowman's membrane with irregular collagen, posterior aggregates of amorphous material, and focal endothelial degeneration. The fifth patient had graft ulceration and vascularization that required removal of the lenticule. She underwent a penetrating keratoplasty five months later and histopathologic examination demonstrated persistent folds in Descemet's membrane. Immunostaining of specimens from three cases disclosed a reduced expression of sulfated epitopes of keratan sulfate and an increase in sulfated dermatan sulfate in the lenticule and host corneal tissues. These alterations in stromal proteoglycans are characteristic of stromal scars and keratoconus and provide evidence of pathologic processes in the graft tissue. Because of potential complications, epikeratoplasty should be considered only for those patients who are unsuitable candidates for contact lenses or penetrating keratoplasty.

Topics: Adult; Aged; Aged, 80 and over; Cicatrix; Cornea; Corneal Stroma; Corneal Transplantation; Dermatan Sulfate; Female; Fibrosis; Humans; Keratan Sulfate; Keratoconus; Male; Middle Aged; Postoperative Complications; Reoperation; Visual Acuity

Human corneas from normal (healthy) donors and patients with keratoconus were either metabolically labelled under organ culture conditions or investigated without preincubation. The sulfated proteoglycans were isolated from a 4 M guanidinium chloride/2% Triton X 100 extract. Two predominant proteoglycans were obtained from normal cornea after digestion of total sulfated proteoglycans with chondroitin ABC-lyase or endo-beta-galactosidase. One had an overall mass of 150 kDa, two dermatan sulfate chains (Mr approximately 50 kDa) with an iduronic acid content of 24%-28% and, after chondroitin ABC-lyase digestion, a core protein of 48 kDa. The other proteoglycan had an overall mass of 110 kDa, one keratan sulfate chain of approximately 60 kDa and, following endo-beta-galactosidase (keratanase) digestion, a core protein of 46 kDa. Each proteoglycan population was further fractionated into two subpopulations by chromatography on concanavalin A-Sepharose. The dermatan sulfate- and keratan sulfate-containing proteoglycans isolated from keratan and healthy cornea had comparable Mr values and core proteins with identical molecular weights, but the ratio of dermatan sulfate/keratan sulfate proteoglycan was increased in keratoconic cornea and the keratan sulfate chains of two keratan sulfate proteoglycans from keratoconic cornea were considerably shorter (Mr 44 and 33 kDa) than those from normal corneas.

Topics: Adult; beta-Galactosidase; Chondroitin Lyases; Chondroitin Sulfate Proteoglycans; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cornea; Culture Techniques; Dermatan Sulfate; Electrophoresis, Polyacrylamide Gel; Glycosaminoglycans; Humans; Keratan Sulfate; Keratoconus; Keratoplasty, Penetrating; Middle Aged; Molecular Weight; Proteoglycans

Monoclonal antibody against keratan sulfate (KS) was used for immunofluorescent staining of sections of human corneas from 8 normal eyes, 19 with keratoconus, 4 with pellucid marginal degeneration, 5 with primary macular corneal dystrophy, and 1 with recurrent macular corneal dystrophy. The anti-KS monoclonal antibody did not stain the corneas with primary macular corneal dystrophy, but stained all other corneas to varying degrees. Staining intensity was weaker than normal in most keratoconus and pellucid marginal degeneration corneas, and was very weak in a case of macular corneal dystrophy that had recurred in a transplanted normal cornea. In several corneas with keratoconus, normal staining was seen at the periphery, and staining intensity decreased in the thinned central portion of the stroma. The decreased KS staining was not localized in stromal scar tissue found in the keratoconus and pellucid marginal degeneration corneas. Quantitation of relative staining intensity found keratoconus and pellucid marginal degeneration corneas to be 49% and 40% as intensely stained, respectively, as normal corneas, a statistically significant decrease (P less than 0.01). Distribution of staining intensities of the keratoconus corneas demonstrated a single modality. These results are in agreement with findings of previous biochemical studies, which show reduction of highly sulfated keratan sulfate epitopes in corneas from keratoconus and pellucid marginal degeneration, and absence of sulfated keratan sulfate epitopes in macular corneal dystrophy.

Topics: Antibodies, Monoclonal; Chondroitin Sulfate Proteoglycans; Corneal Diseases; Corneal Dystrophies, Hereditary; Fluorescent Antibody Technique; Glycosaminoglycans; Humans; Keratan Sulfate; Keratoconus; Lumican; Proteoglycans

Extracts of normal and keratoconus human corneas were assayed for keratan sulfate (KS) and for the protein core of corneal keratan sulfate proteoglycan (KSPG), using a solid-phase immunoassay. Aliquots from guanidine-HCl extracts of four to six pooled corneas were adsorbed to nitrocellulose membrane and assayed for binding of a monoclonal antibody against KS or of an affinity-purified rabbit polyclonal antibody against the KSPG protein core using 125I-labeled secondary antibodies. The amount of antigen in the extracts was estimated from standard curves with purified bovine KSPG. Extracts from keratoconus corneas contained only an average of 48% as much (P less than 0.001) KS-antigen as normal corneal extracts when assayed with anti-KS monoclonal antibody. There was no significant difference in the amount of KSPG core protein antigens in keratoconus and normal corneal extracts. These results suggest that corneas with keratoconus contain a form of KSPG that contains fewer keratan sulfate chains or in which the keratan sulfate has a modified structure.

Topics: Antibodies, Monoclonal; Chondroitin Sulfate Proteoglycans; Cornea; Epitopes; Glycosaminoglycans; Humans; Immunoassay; Keratan Sulfate; Keratoconus; Lumican

Topics: Animals; Autoradiography; Chondroitin Sulfates; Cornea; Culture Techniques; Dermatan Sulfate; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Keratan Sulfate; Keratitis; Keratoconus; Rabbits; Time Factors