Corneal keratocytes: phenotypic and species differences in abundant protein expression and in vitro light-scattering.
OBJECTIVE
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.
Jester
James V
JV
Department of Ophthalmology, University of California at Irvine, Irvine, California 92868, USA. jjester@uci.edu
Budge
Abhijit
A
Fisher
Steven
S
Huang
Jiying
J
D000818:Animals; D015153:Blotting, Western; D002645:Chickens; D003319:Corneal Stroma / Q000166:cytology*; D004591:Electrophoresis, Polyacrylamide Gel; D019573:Epithelium, Corneal / Q000378:metabolism / Q000528:radiation effects*; D005136:Eye Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism* / Q000528:radiation effects*; D006801:Humans; D008027:Light; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010449:Peptide Mapping; D010641:Phenotype; D011817:Rabbits; D012542:Scattering, Radiation; D013045:Species Specificity; D013552:Swine
D005136:Eye Proteins