title: Myofibroblast differentiation modulates keratocyte crystallin protein expression, concentration, and cellular light scattering.
abstract: OBJECTIVE
  The purpose of this study was to determine whether myofibroblast differentiation altered keratocyte crystallin protein concentration and increased cellular light scattering.
  
  
  METHODS
  Serum-free cultured rabbit corneal keratocytes and TGFβ (5 ng/mL) induced myofibroblasts were harvested and counted and protein/RNA extracted. Expression of myofibroblast and keratocyte markers was determined by real-time PCR and Western blot analysis. The cell volume of calcein AM-loaded keratocytes and myofibroblasts was determined by using nonlinear optical microscopy. Cellular light scattering of transformed myofibroblasts expressing human keratocyte crystallins was measured by reflectance confocal microscopy.
  
  
  RESULTS
  Differentiated myofibroblasts showed a significant decrease in RNA levels for the keratocyte markers ALDH1A1, lumican, and keratocan and a significant increase in the myofibroblast marker α-smooth muscle actin. Volumetric and protein measurements showed that myofibroblast differentiation significantly increased cytoplasmic volume (293%; P < 0.001) and water-soluble and -insoluble protein content per cell (respectively, 442% and 431%; P < 0.002) compared to keratocytes. Western blot analysis showed that the level of ALDH1A1 protein per cell was similar between myofibroblasts and keratocytes, but was substantially reduced as a percentage of total water-soluble protein. Light scattering measurements showed that induced expression of corneal crystallins significantly decreased light scattering.
  
  
  CONCLUSIONS
  These data suggest that myofibroblast differentiation leads to a marked increase in cell volume and dilution of corneal crystallins associated with an increase in cellular light scattering.
authors.affiliation: Gavin Herbert Eye Institute, University of California, Irvine, Irvine, California 92697, USA. jjester@uci.edu
authors.forename: James V
authors.initials: JV
authors.lastname: Jester
authors.forename: Donald
authors.initials: D
authors.lastname: Brown
authors.forename: Aglaia
authors.initials: A
authors.lastname: Pappa
authors.forename: Vasilis
authors.initials: V
authors.lastname: Vasiliou
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D060527:Corneal Keratocytes / Q000166:cytology / Q000378:metabolism*; D003319:Corneal Stroma / Q000166:cytology* / Q000378:metabolism; D003459:Crystallins / Q000096:biosynthesis / Q000235:genetics*; D016895:Culture Media, Serum-Free; D008027:Light; D018613:Microscopy, Confocal; D058628:Myofibroblasts / Q000166:cytology* / Q000378:metabolism; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction; D012542:Scattering, Radiation
chemical_list: D003459:Crystallins; D016895:Culture Media, Serum-Free