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J. Biol. Chem., Vol. 278, Issue 46, 45629-45637, November 14, 2003

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Keratocyte Phenotype Mediates Proteoglycan Structure

A ROLE FOR FIBROBLASTS IN CORNEAL FIBROSIS^*

James L. Funderburgh, Mary M. Mann, and Martha L. Funderburgh

From the Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213–2588

In pathological corneas, accumulation of fibrotic extracellular matrix is characterized by proteoglycans with altered glycosaminoglycans that contribute to the reduced transparency of scarred tissue. During wound healing, keratocytes in the corneal stroma transdifferentiate into fibroblasts and myofibroblasts. In this study, molecular markers were developed to identify keratocyte, fibroblast, and myofibroblast phenotypes in primary cultures of corneal stromal cells and the structure of glycosaminoglycans secreted by these cells was characterized. Quiescent primary keratocytes expressed abundant protein and mRNA for keratocan and aldehyde dehydrogenase class 3 and secreted proteoglycans containing macromolecular keratan sulfate. Expression of these marker compounds was reduced in fibroblasts and also in transforming growth factor--induced myofibroblasts, which expressed high levels of -smooth muscle actin, biglycan, and the extra domain A (EDA or EIIIA) form of cellular fibronectin. Collagen types I and III mRNAs were elevated in both fibroblasts and in myofibroblasts. Expression of these molecular markers clearly distinguishes the phenotypic states of stromal cells in vitro. Glycosaminoglycans secreted by fibroblasts and myofibroblasts were qualitatively similar to and differed from those of keratocytes. Chondroitin/dermatan sulfate abundance, chain length, and sulfation were increased as keratocytes became fibroblasts and myofibroblasts. Fluorophore-assisted carbohydrate electrophoresis analysis demonstrated increased N-acetylgalactosamine sulfation at both 4- and 6-carbons. Hyaluronan, absent in keratocytes, was secreted by fibroblasts and myofibroblasts. Keratan sulfate biosynthesis, chain length, and sulfation were significantly reduced in both fibroblasts and myofibroblasts. The qualitatively similar expression of glycosaminoglycans shared by fibroblasts and myofibroblasts suggests a role for fibroblasts in deposition of non-transparent fibrotic tissue in pathological corneas.

Received for publication, March 31, 2003 , and in revised form, August 1, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY221633.

^* This work was supported by National Institutes of Health Grants EY09368 (to J. L. F.), EY003263 (to N. S.), and 30-EY08098 (University of Pittsburgh, Department of Ophthalmology Core Grant), Research to Prevent Blindness, and Eye and Ear Foundation of Pittsburgh. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A Jules and Doris Stein Research to Prevent Blindness Professor. To whom correspondence should be addressed: Dept. of Ophthalmology, University of Pittsburgh, 1011 Eye and Ear Institute, 203 Lothrop St., Pittsburgh, PA 15213-2588. Tel.: 412-647-3853; Fax: 412-647-5880; E-mail: jlfunder{at}pitt.edu.

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