Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes

Pei, Ying; Reins, Rose Y.; McDermott, Alison M.

doi:10.1016/j.exer.2006.05.011

Cited by 73 publications

(70 citation statements)

References 36 publications

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“…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).…”

supporting

confidence: 71%

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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supporting

confidence: 71%

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

et al. 2008

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“…All have previously been utilized to demonstrate the activated repair subtypes of corneal stromal cells. 8,20,22 All of these markers were positively detected in the 2D-activated stromal cells (Fig. 4O, T and Y) before 3D hydrogel encapsulation.…”

Section: Resultsmentioning

confidence: 96%

“…Previous studies have demonstrated that expression of these markers is not detected in activated, stromal cell repair subtypes in situ or after culture in serum. 22 All three keratocyte markers were positively expressed in explant, transwell, and conditioned media co-cultured stromal cells (Fig. 4B-D, F-H, K-M); however, the level of fluorescence for all the markers appeared lower in the cells cultured in conditioned media (Fig.…”

Section: Resultsmentioning

confidence: 99%

Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model

Wilson

Yang²,

Haj³

2014

Tissue Engineering Part A

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In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. Co-culturing aims at recapturing this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. Corneal stromal cells were activated to their injury phenotype (fibroblasts) and expanded before being encapsulated in type I collagen hydrogels constructs. Three different epithelial-stromal co-culture methods were then examined: epithelial explant; transwell; and the use of conditioned media. The aim was to determine whether the native, inactivated keratocyte cell phenotype could be restored in vitro. Media supplementation with transforming growth factor beta-1 (TGF-b1) was then used to determine whether the inactivated stromal cells retained their plasticity in vitro and could be re-activated to the fibroblast phenotype. Finally, media supplementation with wortmannin was used to inhibit epithelial-stromal cell interactions. Two different nondestructive techniques, spherical indentation and optical coherence tomography, were used to reveal how epithelial-stromal co-culturing with TGF-b1, and wortmannin media supplementation, respectively, affect stromal cell behavior and differentiation in terms of construct contraction and elastic modulus measurement. Cell viability, phenotype, morphology, and protein expression were investigated to corroborate our mechanical findings. It was shown that activated stromal cells could be inactivated to a keratocyte phenotype via co-culturing and that they retained their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a nonactivated keratocyte cell lineage in terms of behavior and biological properties; and then back again via TGF-b1 media supplementation. It was then revealed that epithelial-stromal interactions can be blocked via the use of wortmannin inhibition. A greater understanding of stromal-epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury by which such interactions are vital.

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“…The expression of corneal crystallins in keratocytes has been studied and shown to be substantially decreased by culture in serum-or growth factor-supplemented medium. 22,26,[35][36][37] Studies of TKT, an abundantly expressed mammalian corneal crystallin, have also established that the transition of keratocytes to serum-cultured, repair fibroblasts involves degradation of TKT by the ubiquitinproteasome pathway. 36 However, there has been conflicting evidence regarding the expression of TKT in cultured myofibroblast, with one report suggesting that corneal crystallin protein expression is uncoupled from TGF␤-induced myofibroblast differentiation.…”

Section: Discussionmentioning

confidence: 99%

“…18,26 Furthermore, woundhealing fibroblasts and myofibroblasts show a marked increase in light scattering or corneal haze, as detected by in vivo confocal reflectance microscopy. 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.…”

mentioning

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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Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes

Cited by 73 publications

References 36 publications

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

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

Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model

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

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