Myofibroblast differentiation of normal human keratocytes and hTERT, extended-life human corneal fibroblasts

James V. Jester, Jiying Huang, Stephen Fisher, Jennifer Spiekerman, Jin Ho Chang, Woodring E. Wright, Jerry W. Shay

Research output: Contribution to journalArticle

93 Citations (Scopus)

Abstract

PURPOSE. The purpose of this study was to determine whether TGFβ induces myofibroblast differentiation in cultured human keratocytes and in telomerase (hTERT)-immortalized human corneal fibroblast cell lines. METHODS. Normal human corneal keratocytes were isolated from donor corneas of various ages and grown under serum-free (cultured keratocytes) or serum-added (corneal fibroblasts) conditions. Corneal fibroblasts were infected with the MPSV-hTERT retroviral vector, and selected clones were isolated and characterized by chromosomal karyotyping. The responses of normal cultured keratocytes and serum-starved corneal fibroblasts to TGFβ in the presence or absence of Arg-Gly-Asp (RGD)-containing peptides and neutralizing antibodies to platelet-derived growth factor (PDGF) were characterized by immunocytochemistry, Western blot analysis, and real-time PCR, to identify assembly of actin filaments, formation of focal adhesions, and expression of α-smooth muscle actin (α-SMA). RESULTS. Treatment of cultured keratocytes with TGFβ (1 ng/mL) induced cell spreading, assembly of actin filaments, formation of focal adhesions, and expression of α-SMA, which was blocked by the addition of RGD-containing peptides (100 μM). A similar response was identified in hTERT-expressing human corneal fibroblast cell lines, showing a 69-fold increase in a-SMA message. Furthermore, treatment of hTERT corneal fibroblasts with RGD or anti-PDGF inhibited myofibroblast differentiation. Karyotype analysis of hTERT corneal fibroblasts identified age-dependent chromosomal aberrations in cells of older donors but not in those of a 10-year-old donor. CONCLUSIONS. Induction of myofibroblast differentiation by TGFβ 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.

Original languageEnglish (US)
Pages (from-to)1850-1858
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number5
DOIs
StatePublished - May 1 2003

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Myofibroblasts
Fibroblasts
Platelet-Derived Growth Factor
Corneal Keratocytes
Focal Adhesions
Actin Cytoskeleton
Serum
Cell Line
Karyotyping
Telomerase
Neutralizing Antibodies
Karyotype
Chromosome Aberrations
Cornea
Smooth Muscle
Actins
Real-Time Polymerase Chain Reaction
Signal Transduction
Clone Cells
Western Blotting

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Myofibroblast differentiation of normal human keratocytes and hTERT, extended-life human corneal fibroblasts. / Jester, James V.; Huang, Jiying; Fisher, Stephen; Spiekerman, Jennifer; Chang, Jin Ho; Wright, Woodring E.; Shay, Jerry W.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 5, 01.05.2003, p. 1850-1858.

Research output: Contribution to journalArticle

Jester, James V. ; Huang, Jiying ; Fisher, Stephen ; Spiekerman, Jennifer ; Chang, Jin Ho ; Wright, Woodring E. ; Shay, Jerry W. / Myofibroblast differentiation of normal human keratocytes and hTERT, extended-life human corneal fibroblasts. In: Investigative Ophthalmology and Visual Science. 2003 ; Vol. 44, No. 5. pp. 1850-1858.
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T1 - Myofibroblast differentiation of normal human keratocytes and hTERT, extended-life human corneal fibroblasts

AU - Jester, James V.

AU - Huang, Jiying

AU - Fisher, Stephen

AU - Spiekerman, Jennifer

AU - Chang, Jin Ho

AU - Wright, Woodring E.

AU - Shay, Jerry W.

PY - 2003/5/1

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N2 - PURPOSE. The purpose of this study was to determine whether TGFβ induces myofibroblast differentiation in cultured human keratocytes and in telomerase (hTERT)-immortalized human corneal fibroblast cell lines. METHODS. Normal human corneal keratocytes were isolated from donor corneas of various ages and grown under serum-free (cultured keratocytes) or serum-added (corneal fibroblasts) conditions. Corneal fibroblasts were infected with the MPSV-hTERT retroviral vector, and selected clones were isolated and characterized by chromosomal karyotyping. The responses of normal cultured keratocytes and serum-starved corneal fibroblasts to TGFβ in the presence or absence of Arg-Gly-Asp (RGD)-containing peptides and neutralizing antibodies to platelet-derived growth factor (PDGF) were characterized by immunocytochemistry, Western blot analysis, and real-time PCR, to identify assembly of actin filaments, formation of focal adhesions, and expression of α-smooth muscle actin (α-SMA). RESULTS. Treatment of cultured keratocytes with TGFβ (1 ng/mL) induced cell spreading, assembly of actin filaments, formation of focal adhesions, and expression of α-SMA, which was blocked by the addition of RGD-containing peptides (100 μM). A similar response was identified in hTERT-expressing human corneal fibroblast cell lines, showing a 69-fold increase in a-SMA message. Furthermore, treatment of hTERT corneal fibroblasts with RGD or anti-PDGF inhibited myofibroblast differentiation. Karyotype analysis of hTERT corneal fibroblasts identified age-dependent chromosomal aberrations in cells of older donors but not in those of a 10-year-old donor. CONCLUSIONS. Induction of myofibroblast differentiation by TGFβ 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.

AB - PURPOSE. The purpose of this study was to determine whether TGFβ induces myofibroblast differentiation in cultured human keratocytes and in telomerase (hTERT)-immortalized human corneal fibroblast cell lines. METHODS. Normal human corneal keratocytes were isolated from donor corneas of various ages and grown under serum-free (cultured keratocytes) or serum-added (corneal fibroblasts) conditions. Corneal fibroblasts were infected with the MPSV-hTERT retroviral vector, and selected clones were isolated and characterized by chromosomal karyotyping. The responses of normal cultured keratocytes and serum-starved corneal fibroblasts to TGFβ in the presence or absence of Arg-Gly-Asp (RGD)-containing peptides and neutralizing antibodies to platelet-derived growth factor (PDGF) were characterized by immunocytochemistry, Western blot analysis, and real-time PCR, to identify assembly of actin filaments, formation of focal adhesions, and expression of α-smooth muscle actin (α-SMA). RESULTS. Treatment of cultured keratocytes with TGFβ (1 ng/mL) induced cell spreading, assembly of actin filaments, formation of focal adhesions, and expression of α-SMA, which was blocked by the addition of RGD-containing peptides (100 μM). A similar response was identified in hTERT-expressing human corneal fibroblast cell lines, showing a 69-fold increase in a-SMA message. Furthermore, treatment of hTERT corneal fibroblasts with RGD or anti-PDGF inhibited myofibroblast differentiation. Karyotype analysis of hTERT corneal fibroblasts identified age-dependent chromosomal aberrations in cells of older donors but not in those of a 10-year-old donor. CONCLUSIONS. Induction of myofibroblast differentiation by TGFβ 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.

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