Effects of Ultraviolet-A and Riboflavin on the Interaction of Collagen and Proteoglycans during Corneal Cross-linking

Abstract

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen ␣ and ␤ chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.The cornea is the transparent, dome-shaped tissue covering the front of the eye. It is a powerful refracting surface, providing 65-75% of the eye's focusing power, and is made up of three distinct layers: epithelium, stroma, and endothelium. The stroma comprises about 90% of cornea thickness in humans (1).Although it is very highly innervated, it does not contain any blood vessels (2-4). Collagen gives the cornea its strength, elasticity, and form (5). The unique molecular shape, paracrystalline arrangement, and very regular fine diameter of the evenly spaced collagen fibrils are essential in producing a transparent cornea (6, 7).In the corneal stromal extracellular matrix, glycosaminoglycan (GAG) 2 polysaccharides are the most abundant negatively charged macromolecules and are classified on the basis of their repeating disaccharide structures into four main groups: keratan sulfate (KS), chondroitin sulfate/dermatan sulfate (CS/DS), heparan sulfate and heparin, and hyaluronan. Glycosaminoglycans, normally covalently attached to proteoglycan (PG) core proteins, play important roles in corneal transparency, nerve growth cone guidance, and cell adhesion, largely dependent on their patterns of sulfation or lack of it (8, 9). The corneal stroma is comp...

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