Format

Send to:

Choose Destination
See comment in PubMed Commons below
Invest Ophthalmol Vis Sci. 2012 Feb 16;53(2):770-8. doi: 10.1167/iovs.11-9092.

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

Author information

  • 1Gavin Herbert Eye Institute, University of California, Irvine, Irvine, California 92697, USA. jjester@uci.edu

Abstract

PURPOSE:

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.

PMID:
22247459
[PubMed - indexed for MEDLINE]
PMCID:
PMC3317419
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Silverchair Information Systems Icon for PubMed Central
    Loading ...
    Write to the Help Desk