Corneal Keratocytes: Phenotypic and Species Differences in Abundant Protein Expression and In Vitro Light-Scattering

Jester, James V.; Budge, Abhijit; Fisher, Steven A.; Huang, Jingjing

doi:10.1167/iovs.04-1225

Cited by 109 publications

(128 citation statements)

References 30 publications

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“…The corneal myofibroblast phenotype present during wound healing is characterized by the expression of ␣-smooth muscle actin (␣-SMA), a filamentous protein that plays a role in the matrix contraction (9). Cultured rabbit corneal myofibroblasts scatter more light than keratocytes and exhibit reduced expression of both TKT and ALDH1 (16). A recent study in human cells corroborates the rabbit studies: ALDH3 and TKT are both reduced in repair fibroblast and myofibroblast populations compared with the quiescent keratocyte (17).…”

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confidence: 72%

The Development of a Tissue-Engineered Cornea: Biomaterials and Culture Methods

et al. 2008

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ABSTRACT:The field of corneal tissue engineering has made many strides in recent years. The challenges of engineering a biocompatible, mechanically stable, and optically transparent tissue are significant. To overcome these challenges, researchers have adopted two basic approaches: cell-based strategies for manipulating cells to create their own extracellular matrix, and scaffold-based strategies for providing strong and transparent matrices upon which to grow cells. Both strategies have met with some degree of success. In addition, recent advances have been made in innervating a tissueengineered construct. Future work will need to focus on further improving mechanical stability of engineered constructs as well as improving the host response to implantation.

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confidence: 72%

The Development of a Tissue-Engineered Cornea: Biomaterials and Culture Methods

et al. 2008

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“…Additional study is needed to determine what factors are most important. We speculate that differences in the timing of myofibroblast appearance in the wounded corneas of different species are due to differences in marker protein expression, such as alpha smooth muscle actin, in the stroma of different species, based on the study performed by Jester and his coworkers (Jester et al 2005) that showed human, mouse, rabbit, chicken and pig corneal keratocytes have diverse protein expression patterns.…”

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confidence: 99%

A novel method for generating corneal haze in anterior stroma of the mouse eye with the excimer laser

Mohan

Stapleton

Sinha

et al. 2008

Experimental Eye Research

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Refractive surgery is a popular method used to reduce or eliminate dependence on glasses and contact lenses. Corneal haze is one of the common complications observed after photorefractive keratectmomy (PRK). The objective of this study was to develop an in vivo mouse model that consistently produces moderate to severe corneal haze in the anterior stroma of the mouse cornea after excimer laser treatment to study myofibroblast biology and corneal wound healing in a genetically defined model. Regular-or irregular-phototherapeutic keratectomy (PTK) was performed on black C57BL/6 mice with the Summit Apex excimer laser (Alcon, Ft. Worth, TX). Different numbers of laser pulses (45; ablation depth ~10 μm) were fired on the central cornea, after scraping the epithelium prior to excimer laser ablation. Irregularity was generated by positioning a fine mesh screen in the path of laser after firing 50% of the pulses. Eyes were collected 1, 2, 3 or 4 weeks after the procedure. Haze formation was gauged with slit lamp biomicroscopy. Immunocytochemistry was used to determine number of myofibroblasts in the mouse cornea using antibodies specific for the myofibroblast marker alpha-smooth muscle actin (SMA). The numbers of SMA-positive cells/400X microscopic were determined by counting within the stroma. Statistical analysis was performed using analysis of variance (AVOVA) with the Bonferonni-Dunn adjustment for repeated measures. Regular-PTK with epithelial scrape (Group 3) and irregular-PTK with epithelial scrape (Group 4) in the mouse eyes were performed to produce corneal haze. Eyes collected 4 weeks after regular-or irregular-PTK after epithelial scrape showed 22 ± 6.6 (Group 3) or 34 ± 7.9 (Group 4) SMA-positive cells in the anterior cornea. The difference in the SMA-positive cells detected among the groups was statistically significant (p < 0.01). Less than 4 SMA-positive cells were detected in the tissue sections of the mouse eyes collected after 1, 2 or 3 weeks of regular (Group 3) or irregular PTK (Group 4) or controls (Group 1 and 2). The optimized PTK excimer laser conditions developed in this study produces haze selectively in anterior stroma of the mouse cornea immediately beneath the epithelial basement membrane. Irregular PTK performed after epithelial scrape by applying 45 laser pulses was found to be the most effective method to generate myofibroblasts. This PTK technique for inducing haze in mouse cornea in vivo provides a useful model for studying wound healing and myofibroblast biology in transgenic mice.

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“…10,27,28 Growth factor-stimulated rabbit keratocytes in culture also show significantly reduced levels of crystallin protein and increased cellular light scattering, with TGF␤-induced myofibroblasts showing the greatest changes. 22 Although expression of corneal crystallins has been shown to correlate with cellular light scattering and corneal haze, the molecular and biophysical mechanisms affecting cellular transparency have yet to be explained. In this study, we evaluated the biophysical characteristics of keratocytes and myofibroblasts on the basis of cell size, protein content, expression of corneal crystallins, and changes in light scattering after induced corneal crystallin expression.…”

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confidence: 99%

Myofibroblast Differentiation Modulates Keratocyte Crystallin Protein Expression, Concentration, and Cellular Light Scattering

Jester

Brown

Pappa

et al. 2012

Invest. Ophthalmol. Vis. Sci.

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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 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 AMloaded 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 ␣-smooth muscle actin. Volumetric and protein measurements showed that myofibroblast differentiation significantly increased cytoplasmic volume (293%; P Ͻ 0.001) and water-soluble and -insoluble protein content per cell (respectively, 442% and 431%; P Ͻ 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. (Invest Ophthalmol Vis Sci. 2012;53:770 -778) DOI: 10.1167/iovs.11-9092 P revious studies have shown that differentiation of keratocytes into myofibroblasts is regulated in part by transforming growth factor (TGF)-␤.1,2 Under serum-free culture conditions, rabbit, bovine, and human corneal keratocytes maintain a quiescent phenotype, dendritic morphology, and high expression of phenotypic markers, including keratocan, lumican, and the corneal crystallins, aldehyde dehydrogenase isozymes 3A1 and 1A1 (ALDH3A1/1A1) and transketolase (TKT). 3-5Treatment of cultured keratocytes with TGF␤ leads to cell spreading, actin filament assembly, and downregulated expression of keratocyte-specific genes that is coupled with the de novo upregulated expression of ␣-smooth muscle actin (␣-SMA), the phenotypic marker for myofibroblast differentiation. 6 Myofibroblasts play a critical role in corneal wound healing that involves deposition and organization of extracellular matrix leading to wound contraction. [7][8][9] Neutralizing antibodies to TGF␤ have been shown to block the appearance of myofibroblasts in corneal wounds and significantly reduce corneal scarring and the development of corneal haze. 10,11The loss of transparency after corneal injury and scarring has long been associated with the deposition of abnormally arranged and disorg...

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Corneal Keratocytes: Phenotypic and Species Differences in Abundant Protein Expression and In Vitro Light-Scattering

Cited by 109 publications

References 30 publications

The Development of a Tissue-Engineered Cornea: Biomaterials and Culture Methods

The Development of a Tissue-Engineered Cornea: Biomaterials and Culture Methods

A novel method for generating corneal haze in anterior stroma of the mouse eye with the excimer laser

Myofibroblast Differentiation Modulates Keratocyte Crystallin Protein Expression, Concentration, and Cellular Light Scattering

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