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(Investigative Ophthalmology and Visual Science. 2005;46:2369-2378.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-1225
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Corneal Keratocytes: Phenotypic and Species Differences in Abundant Protein Expression and In Vitro Light-Scattering

James V. Jester,1 Abhijit Budge,2 Steven Fisher,2 and Jiying Huang2

1From the Department of Ophthalmology, University of California at Irvine, Irvine, California; and the 2Departament of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas.

PURPOSE. Previous studies suggest that corneal haze after injury involves changes in the light-scattering properties of keratocytes that are possibly linked to the abundant expression of water-soluble proteins. The purpose of this study was to determine the protein expression pattern of keratocytes from different species and different cultured rabbit keratocyte phenotypes and to assess differences in light-scattering in vitro.

METHODS. Water-soluble proteins were isolated from corneal epithelial cells and keratocytes of several species, including human (Hu), mouse (Mo), rabbit (Ra), chicken (Ch), and pig (P) and different cultured rabbit keratocyte phenotypes. Proteins were then characterized by SDS-PAGE, tryptic peptide sequence analysis, and Western blot analysis. Light-scattering and actin organization from cultured cells were determined with confocal reflectance and fluorescence microscopy, respectively.

RESULTS. Protein expression patterns varied substantially between species and cell types, with five new abundantly expressed proteins identified including, LDH (Ra, Ch), G3PDH (Hu, Ch), pyruvate kinase (Ch), Annexin II (Ch), and protein disulfide isomerase (Ch). Different rabbit keratocyte phenotypes also showed different levels of expression of ALDH1A1 and TKT, with myofibroblasts showing the greatest reduction. Myofibroblasts showed significantly greater (P < 0.05) light-scattering but also showed the greatest organization of actin filaments.

CONCLUSIONS. Abundant protein expression is a characteristic feature of corneal keratocytes that is lost when cells are phenotypically modulated in culture. Greater light-scattering by myofibroblasts also provides support for a link between cellular transparency and haze after injury that is possibly related to loss of protein expression or development of prominent actin filament bundles.




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