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J.L. Funderburgh, M.M. Mann, M.L. Funderburgh; Biosynthesis of Corneal Dermatan Sulfate: Are Myofibroblasts the Source of Stromal Fibrosis ? . Invest. Ophthalmol. Vis. Sci. 2003;44(13):878.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose In early stages of corneal wound healing keratocytes become mitotic and exhibit cytoskeletal characteristics of fibroblasts. Later, myofibroblasts containing smooth muscle actin (SMA) appear in response to transforming growth factor beta (TGFß). We showed that coincident with SMA, TGFß induces secretion of fibrosis-associated matrix molecules including collagen I and III, biglycan, and cellular fibronectin (cFN) in keratocytes (J Biol Chem 276, 44173). Corneal scars contain dermatan sulfate (DS) of increased size and sulfation. This DS is implicated in altered stromal hydration and collagen organization in the pathological stroma. This study asks if altered DS is a product of myofibroblasts similar to other recognized fibrotic components. Methods Primary bovine keratocytes became fibroblastic after 5-6 days in 2% fetal bovine serum or myofibroblastic in 2% serum and 2 ng/ml TGFß1. Cell phenotype was characterized using immunoblotting and real-time PCR for protein and mRNA pools of aldehyde dehydrogenase, keratocan, SMA, biglycan, cFN, collagen I and III and by immunohistology of cytoskeletal components. The size, sulfation, and composition of dermatan sulfate were examined by gel electrophoresis of sulfate-labeled DS and by fluorophore assisted carbohydrate electrophoresis (FACE). Results When keratocytes became fibroblasts they lost expression of aldehyde dehydrogenase and keratocan mRNA, developed actin stress fibers and focal adhesions. In TGFß the cells also expressed SMA, cFN, and biglycan protein and mRNA. These markers provided a clear distinction between keratocyte, fibroblast, and myofibroblast phenotypes. DS was markedly increased in amount, size, and sulfation in fibroblasts compared to keratocytes. Both 4-O and 6-O-sulfation was increased and the unsulfated component of DS was reduced. Qualitatively similar changes in DS occurred in myofibroblasts but the alterations were more pronounced. Conclusions Secretion of some matrix components associated with corneal fibrosis can be clearly associated with the myofibroblastic phenotype, however both fibroblasts and myofibroblasts secrete increased amounts of DS with altered structure. These results demonstrate that long-term alterations stromal matrix associated with stromal scarring are not all TGFß-related and that fibroblasts participate in the fibrotic as well as remodeling phases of wound healing.
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