Differential Relative Sulfation of Keratan Sulfate Glycosaminoglycan in the Chick Cornea during Embryonic Development

Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Cardiff, Wales, United Kingdom.
Investigative ophthalmology & visual science (Impact Factor: 3.4). 10/2009; 51(3):1365-72. DOI: 10.1167/iovs.09-4004
Source: PubMed


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.
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.
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.
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.

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    • "Our in vivo studies revealed no change in sulfated keratan sulfate three weeks after wounding, with or without Y-27632 application, as identified by immunohistochemistry with an antibody (5D4), which recognizes a minimally pentasulfated epitope on the keratan sulfate glycosaminoglycan chain [50]. Large proteoglycan filaments, which are seen in the stromal matrix in the center of the healing wound, are similar in character to stained structures that have been reported previously in healing corneas [34,35,49] and in embryonic corneas [51]. In these tissues, they represent oversulfated proteoglycans of the chondroitin sulfate/dermatan sulfate class [52], and it is likely that that is the case here too, although we did not definitively identify the glycosaminoglycan as chondroitin sulfate/dermatan sulfate by prior lyase digestion. "
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    ABSTRACT: Our study examined the effect of a selective Rho kinase inhibitor, Y-27632, on corneal wound healing and potential stromal scarring after superficial keratectomy. Rabbit keratocytes were induced into myofibroblasts by transforming growth factor β1 (TGFβ1) either with or without Y-27632. Then α-smooth muscle actin (α-SMA) was examined by immunohistochemistry and western blotting, and the contractility of the seeded collagen gels was measured. Y-27632 eye drops (or vehicle only) were administered to eyes after a superficial keratectomy, and the tissue was examined by immunohistochemistry for α-SMA, collagen types I, II, and III, and keratan sulfate. Electron microscopy was conducted with and without histochemical contrasting of sulfated proteoglycans. Spindle-like cells in culture constituted 99.5±1.1% with TGFβ1 stimulation, but 3.5±1.0% after TGFβ1 and Y-27632 treatment (p<0.01, n=6). α-SMA was seen in 4% of TGFβ1-treated cells, but in only 0.3% of cells with Y-27632 added (p<0.01, n=6), which was confirmed by western blotting. Y-27632 also inhibited the TGFβ1-induced contraction of seeded collagen gels. After superficial keratectomies, collagen type I and keratan sulfate were unchanged by Y-27632 application. Collagen type II was not detected in Y-27632 or vehicle-only corneas. With Y-27632 treatment, α-SMA expression increased and the collagen type III signal became in the weaker subepithelial area. Interestingly, bundles of aligned and uniformly spaced collagen fibrils were more prevalent in keratocytes in Y-27632-treated corneas, which is reminiscent of fibripositor-like structures that have been proposed as a mechanism of matrix deposition in embryonic connective tissues. Y-27632 inhibits keratocyte-to-myofibroblast transition, and its topical application after a superficial lamellar keratectomy elicits an altered wound healing response, with evidence of an embryonic-type deposition of collagen fibrils.
    Molecular vision 06/2012; 18:1727-39. · 1.99 Impact Factor
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    ABSTRACT: Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) in the cornea of the eye, where it exists in proteoglycan (PG) form. KS-PGs have long been thought to play a pivotal role in the establishment and maintenance of the array of regularly-spaced and uniformly- thin collagen fibrils which make up the corneal stroma. This characteristic arrangement of fibrils allows light to pass through the cornea. Indeed, perturbations to the synthesis of KS-PG core proteins in genetically altered mice lead to structural matrix alterations and corneal opacification. Similarly, mutations in enzymes responsible for the sulphation of KS-GAG chains are causative for the inherited human disease, macular corneal dystrophy, which is manifested clinically by progressive corneal cloudiness starting in young adulthood.
    Cellular and Molecular Life Sciences CMLS 03/2010; 67(6):891-906. DOI:10.1007/s00018-009-0228-7 · 5.81 Impact Factor
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