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Myofibroblast differentiation modulates keratocyte crystallin protein expression, concentration, and cellular light scattering.

ABSTRACT: The purpose of this study was to determine whether myofibroblast differentiation altered keratocyte crystallin protein concentration and increased cellular light scattering.Serum-free cultured rabbit corneal keratocytes and TGF? (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.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 ?-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.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.

SUBMITTER: Jester JV

PROVIDER: S-EPMC3317419 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Myofibroblast differentiation modulates keratocyte crystallin protein expression, concentration, and cellular light scattering.

Jester James V JV Brown Donald D Pappa Aglaia A Vasiliou Vasilis V

Investigative ophthalmology & visual science 20120216 2

<h4>Purpose</h4>The purpose of this study was to determine whether myofibroblast differentiation altered keratocyte crystallin protein concentration and increased cellular light scattering.<h4>Methods</h4>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 kerat ...[more]

PMID: 22247459

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Dataset Information

Myofibroblast differentiation modulates keratocyte crystallin protein expression, concentration, and cellular light scattering.

Publications

Myofibroblast differentiation modulates keratocyte crystallin protein expression, concentration, and cellular light scattering.

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