Proteoglycans in the corneal stroma and their role in development and pathology

Transmission electron microscopy of a human cornea with excess chondroitin sulphate/dermatan sulphate glycosaminoglycan showed that changes in proteoglycan structure, content and sulphation lead to the formation of abnormally large collagen fibrils. The lack of sulphation of keratin sulphate in MCD also led to abnormally large fibrils, which are present in the deep stromal layers. These findings suggest overlapping roles of the two proteoglycan populations in the corneal stroma with possible feedback mechanisms, too. Taken together, the findings of this thesis indicate the central role played by proteoglycan-collagen interactions in the development and maintenance of properly formed corneal stroma.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Proteoglycans in the corneal stroma and their role in development and pathology

Transmission electron microscopy of a human cornea with excess chondroitin sulphate/dermatan sulphate glycosaminoglycan showed that changes in proteoglycan structure, content and sulphation lead to the formation of abnormally large collagen fibrils. The lack of sulphation of keratin sulphate in MCD also led to abnormally large fibrils, which are present in the deep stromal layers. These findings suggest overlapping roles of the two proteoglycan populations in the corneal stroma with possible feedback mechanisms, too. Taken together, the findings of this thesis indicate the central role played by proteoglycan-collagen interactions in the development and maintenance of properly formed corneal stroma.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Proteoglycans in the corneal stroma and their role in development and pathology

Transmission electron microscopy of a human cornea with excess chondroitin sulphate/dermatan sulphate glycosaminoglycan showed that changes in proteoglycan structure, content and sulphation lead to the formation of abnormally large collagen fibrils. The lack of sulphation of keratin sulphate in MCD also led to abnormally large fibrils, which are present in the deep stromal layers. These findings suggest overlapping roles of the two proteoglycan populations in the corneal stroma with possible feedback mechanisms, too. Taken together, the findings of this thesis indicate the central role played by proteoglycan-collagen interactions in the development and maintenance of properly formed corneal stroma

Differential relative sulfation of keratan sulfate glycosaminoglycan in the chick cornea during embryonic development

Purpose. To investigate structural remodeling of the developing corneal stroma concomitant with changing sulfation patterns of keratan sulfate (KS) glycosaminoglycan (GAG) epitopes during embryogenesis and the onset of corneal transparency. Methods. Developing chick corneas were obtained from embryonic day (E)12 to E18 of incubation. Extracellular matrix composition and collagen fibril spacing were evaluated by synchrotron x-ray diffraction, hydroxyproline assay, ELISA (with antibodies against lesser and more highly sulfated KS), and transmission electron microscopy with specific proteoglycan staining. Results. A significant relative increase in highly sulfated KS epitope labeling occurred with respect to hydroxyproline content in the final week of chick development, as mean collagen interfibrillar distance decreased. Small KS PG filaments increased in frequency with development and were predominantly fibril associated. Conclusions. The accumulation of highly sulfated KS during the E12 to E18 timeframe could serve to fine tune local matrix hydration and collagen fibril spacing during corneal growth, as gross dehydration and compaction of the stroma progress through the action of the nascent endothelial pump

Large Proteoglycan Complexes and Disturbed Collagen Architecture in the Corneal Extracellular Matrix of Mucopolysaccharidosis Type VII (Sly Syndrome)

Purpose. Deficiencies in enzymes involved in proteoglycan (PG) turnover underlie a number of rare mucopolysaccharidoses (MPS), investigations of which can considerably aid understanding of the roles of PGs in corneal matrix biology. Here, the authors analyze novel pathologic changes in MPS VII (Sly syndrome) to determine the nature of PG-collagen associations in stromal ultrastructure. Methods. Transmission electron microscopy and electron tomography were used to investigate PG-collagen architectures and interactions in a cornea obtained at keratoplasty from a 22-year-old man with MPS VII, which was caused by a compound heterozygous mutation in the GUSB gene. Results. Transmission electron microscopy showed atypical morphology of the epithelial basement membrane and Bowman's layer in MPS VII. Keratocytes were packed with cytoplasmic vacuoles containing abnormal glycosaminoglycan (GAG) material, and collagen fibrils were thinner than in normal cornea and varied considerably throughout anterior (14–32 nm), mid (13–42 nm), and posterior (17–39 nm) regions of the MPS VII stroma. PGs viewed in three dimensions were striking in appearance in that they were significantly larger than PGs in normal cornea and formed highly extended linkages with multiple collagen fibrils. Conclusions. Cellular changes in the MPS VII cornea resemble those in other MPS. However, the wide range of collagen fibril diameters throughout the stroma and the extensive matrix presence of supranormal-sized PG structures appear to be unique features of this disorder. The findings suggest that the accumulation of stromal chondroitin-, dermatan-, and heparan-sulfate glycosaminoglycans in the absence of β-glucuronidase-mediated degradation can modulate collagen fibrillogenesis

Structural Collagen Alterations in Macular Corneal Dystrophy Occur Mainly in the Posterior Stroma

Purpose: Collagen fibrils in the corneal stroma in macular corneal dystrophy, on average, are more closely spaced than in the normal cornea. This study was conducted to investigate if this occurs uniformly across the stroma or is more prevalent at certain stromal depths. Methods: Microbeam synchrotron X-ray fiber diffraction patterns were obtained in 25 μm steps across the whole thickness of a thin strip of a macular corneal dystrophy cornea obtained at keratoplasty. Data were analyzed for mean collagen interfibrillar spacing at all positions. Serum was analyzed immunochemically to determine immunophenotype, and transmission electron microscopy was carried out to visualize stromal ultrastructure. Results: Keratan sulphate was not detectable in blood serum, classifying the disease as macular corneal dystrophy type I. Collagen interfibrillar spacing dropped linearly with stromal depth from the anterior to posterior cornea, measuring 5–10% less in the posterior 100 µm of the MCD stroma compared to the anterior 100 µm (p < 0.001). Isolated pockets of collagen fibrils with unusually large diameters were identified in the deep stroma. Conclusions: Collagen fibril spacing is reduced and large-diameter collagen fibrils are seen in macular corneal dystrophy type I, with the deep stroma affected more. We speculate that the ultrastructural abnormalities are more prevalent in the posterior stroma because the structural influence of sulphated keratan sulphate glycosaminoglycans/proteoglycans is high in this region of the cornea

Large Proteoglycan Complexes and Disturbed Collagen Architecture in the Corneal Extracellular Matrix of Mucopolysaccharidosis Type VII (Sly Syndrome)

Purpose. Deficiencies in enzymes involved in proteoglycan (PG) turnover underlie a number of rare mucopolysaccharidoses (MPS), investigations of which can considerably aid understanding of the roles of PGs in corneal matrix biology. Here, the authors analyze novel pathologic changes in MPS VII (Sly syndrome) to determine the nature of PG-collagen associations in stromal ultrastructure. Methods. Transmission electron microscopy and electron tomography were used to investigate PG-collagen architectures and interactions in a cornea obtained at keratoplasty from a 22-year-old man with MPS VII, which was caused by a compound heterozygous mutation in the GUSB gene. Results. Transmission electron microscopy showed atypical morphology of the epithelial basement membrane and Bowman's layer in MPS VII. Keratocytes were packed with cytoplasmic vacuoles containing abnormal glycosaminoglycan (GAG) material, and collagen fibrils were thinner than in normal cornea and varied considerably throughout anterior (14–32 nm), mid (13–42 nm), and posterior (17–39 nm) regions of the MPS VII stroma. PGs viewed in three dimensions were striking in appearance in that they were significantly larger than PGs in normal cornea and formed highly extended linkages with multiple collagen fibrils. Conclusions. Cellular changes in the MPS VII cornea resemble those in other MPS. However, the wide range of collagen fibril diameters throughout the stroma and the extensive matrix presence of supranormal-sized PG structures appear to be unique features of this disorder. The findings suggest that the accumulation of stromal chondroitin-, dermatan-, and heparan-sulfate glycosaminoglycans in the absence of β-glucuronidase-mediated degradation can modulate collagen fibrillogenesis