Myofibroblast Differentiation of Normal Human Keratocytes and hTERT, Extended-Life Human Corneal Fibroblasts

Jester, James V.; Huang, Jingjing; Fisher, Stephen; Spiekerman, Jennifer; Chang, Jin Ho; Wright, Woodring E.

doi:10.1167/iovs.02-0973

Cited by 128 publications

(104 citation statements)

References 27 publications

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“…Studies were performed using a previously characterized telomerase-infected, extended lifespan human corneal fibroblast cell line, HTK [33]. HTK cells were cultured in serumcontaining (S+) medium consisting of Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO Invitrogen Cell Culture, Carlsbad, CA) supplemented with 1% penicillin, 1% streptomycin and 1% Fungizone (Biowhittaker, Inc., Wakersville, MD) and 10% fetal bovine serum (Sigma Chemical Corp., St. Louis, MO).…”

Section: Cellsmentioning

confidence: 99%

Quantitative assessment of local collagen matrix remodeling in 3-D Culture: The role of Rho kinase

Kim

Lakshman

Petroll

2006

Experimental Cell Research

106

123

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The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling.Human corneal fibroblasts were plated inside 100 μm thick fibrillar collagen matrices and cultured for 24 hours in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph.Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p < 0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01).Overall, the data suggests that Rho kinase dependent contractile force generation leads to coalignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.

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Section: Cellsmentioning

confidence: 99%

Quantitative assessment of local collagen matrix remodeling in 3-D Culture: The role of Rho kinase

Kim

Lakshman

Petroll

2006

Experimental Cell Research

106

123

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“…Human HTCE cells, human HTK immortalized cells (Jester et al, 2003) or corneal stroma fibroblast primary cells were lysed in lysis buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% deoxycholate, 1× protease inhibitor cocktail (Sigma P8340)]. Frozen tissue was homogenized in RIPA buffer containing phosphatase and protease inhibitors.…”

Section: Western Blotting Analysismentioning

confidence: 99%

Wnt/β-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development

et al. 2015

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The development of organs with an epithelial parenchyma relies on reciprocal mesenchymal-epithelial communication. Mouse corneal epithelium stratification is the consequence of a coordinated developmental process based on mesenchymal-epithelial interactions. The molecular mechanism underlying these interactions remains unclear. The Wnt/β-catenin signaling pathway is involved in fundamental aspects of development through the regulation of various growth factors. Here, we show that conditional ablation of either β-catenin (Ctnnb1 cKO ) or co-receptors Lrp5/6 (Lrp5/6 cKO ) in corneal stromal cells results in precocious stratification of the corneal epithelium. By contrast, ectopic expression of a murine Ctnnb1 gainof-function mutant (Ctnnb1 cGOF ) retards corneal epithelium stratification. We also discovered that Bmp4 is upregulated in the absence of β-catenin in keratocytes, which further triggers ERK1/2 (Mapk3/1) and Smad1/5 phosphorylation and enhances transcription factor p63 (Trp63) expression in mouse corneal basal epithelial cells and in a human corneal epithelial cell line (HTCE). Interestingly, mouse neonates given a subconjunctival BMP4 injection displayed a phenotype resembling that of Ctnnb1 cKO . Conditional ablation of Bmp4 eradicates the phenotype produced in Ctnnb1 cKO mice. Furthermore, ChIP and promoter-luciferase assays show that β-catenin binds to and suppresses Bmp4 promoter activity. These data support the concept that cross-talk between the Wnt/β-catenin/ Bmp4 axis (in the stromal mesenchyme) and Bmp4/p63 signaling (in the epithelium) plays a pivotal role in epithelial stratification during corneal morphogenesis.

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“…11,17,18 Although double labeling was not carried out in the present study, we expect that actin filaments and a-SMA would colocalize as they did in a study that reported myofibroblastic transformation in cultures of human corneal stromal keratocytes in the serumfree medium containing TGF-b. 69 Compared with fibroblasts, myofibroblasts are more active in the production of a-SMA, collagen, and fibronectin, and in the formation of organized intracellular actin filaments and focal adhesions, 11 …”

Section: Its Stimulated Organized Collagenous Matrix Secretion and Ormentioning

confidence: 99%

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

Bueno

Saeidi

Melotti

et al. 2009

Tissue Engineering Part A

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The in vitro production of highly organized collagen fibrils by corneal keratocytes in a three-dimensional scaffold-free culture system presents a unique opportunity for the direct observation of organized matrix formation. The objective of this investigation was to develop such a culture system in a glass substrate (for optical accessibility) and to directly examine the effect of reducing serum and=or increasing insulin on the stratification and secretion of aligned matrix by fourth-to fifth-passage bovine corneal stromal keratocytes. Medium concentrations of 0%, 1%, or 10% fetal bovine serum and 0% or 1% insulin-transferrin-selenium were investigated. Highresolution differential interference contrast microscopy, quick-freeze=deep-etch, and conventional transmission electron microscopy were used to monitor the evolution, morphology, and ultrastructure of the cell-matrix constructs. In a medium containing 1% each of serum and insulin-transferrin-selenium, stromal cells stratified and secreted abundant and locally aligned matrix, generating the thickest cell-matrix constructs (allowing handling with forceps). The results of this study have the potential to significantly advance the field of developmental functional engineering of load-bearing tissues by (i) elucidating cues that modulate in vitro cell secretion of organized matrix and (ii) establishing an optically accessible cell culture system for investigating the mechanism of cell secretion of aligned collagen fibrils.

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Myofibroblast Differentiation of Normal Human Keratocytes and hTERT, Extended-Life Human Corneal Fibroblasts

Abstract: Induction of myofibroblast differentiation by TGFbeta in cultured human keratocytes and hTERT corneal fibroblasts occurs through a similar signal transduction pathway to that previously identified in the rabbit, which involves an autocrine PDGF feedback loop.

Cited by 128 publications

References 27 publications

Quantitative assessment of local collagen matrix remodeling in 3-D Culture: The role of Rho kinase

Quantitative assessment of local collagen matrix remodeling in 3-D Culture: The role of Rho kinase

Wnt/β-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

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