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Loss of Alpha3(IV) Collagen Expression Associated with Corneal Keratocyte Activation

¹From the UPMC Eye Center, Ophthalmology and Visual Science Research Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; ²Division of Immunology, Shigei Medical Research Institute, Okayama, Japan; and ³Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio.

PURPOSE. To determine whether changes in the expression of type IV 1, 2, or 3 collagen isoforms are stringently associated with corneal stromal cell activation.

METHODS. Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)–, TGF-ß1–, or fetal bovine serum (FBS)–supplemented DMEM/F12 medium. Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was evaluated by immunocytochemical analysis, Western blot analysis, or both. Concentrations of mRNAs were estimated by quantitative RT-PCR using SYBR Green RT-PCR reagents.

RESULTS. Immunohistochemical analysis indicated that type IV 1, 2, and 3 collagens were expressed in normal rabbit corneal stroma and in keratocytes cultured in serum-free and insulin-supplemented media. However, 3(IV) collagen was not detectable in the regenerating stroma after photorefractive keratectomy (PRK) in rabbit or in corneal stromal cells cultured in media supplemented with FBS, bFGF/HS, or TGF-ß1. 3(IV) collagen mRNA levels were also diminished in the stromal cells cultured in these growth factor-supplemented media. KSPGs (lumican and keratocan) were expressed and secreted in serum-free medium. Although the expression of KSPGs was promoted by insulin, the expression and intracellular levels of lumican and keratocan mRNAs were downregulated by TGF-ß1 and FBS. bFGF/HS promoted the downregulation of intracellular keratocan but not lumican mRNA levels.

CONCLUSIONS. The loss in the expression of 3(IV) collagen is a stringent phenotypic change associated with activation of keratocytes in vivo and in vitro. This phenotypic change in activated corneal stromal cells is induced by bFGF/HS and by TGF-ß1, and it accompanies the downregulation of keratocan expression.