Induction of α-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes

James V. Jester, Patricia A. Barry-Lane, Harrison D Cavanagh, Walter M Petroll

Research output: Contribution to journalArticle

253 Citations (Scopus)

Abstract

The effects of serum, transforming growth factor (TGF)(β1), bFGF, and heparin on in vitro myofibroblast transformation was studied. Primary rabbit corneal keratocytes were grown under serum-free conditions or in media supplemented with serum (10% fetal calf serum), TGF(β1) (0.1-10 ng/ml), basic fibroblast growth factor (bFGF) (0.1-10 ng/ml), or heparin (10 U/ml). Cells were analyzed for expression of α-smooth muscle actin (α-SM actin), α5β1 integrin (the high-affinity fibronectin receptor) and fibronectin by immunoprecipitation, Western blotting, and immunofluorescence. Corneal keratocytes grown in the presence of serum showed a typical fibroblast morphology with induction of α-SM actin expression in 1 to 10% of cells. Addition of bFGF blocked serum-induced α-SM actin expression, whereas addition of TGF(β1) enhanced α-SM actin expression (100%), which in combination with heparin (10 U/ml), led to a pulling apart of the fibroblastic sheet, simulating contraction. Under serum-free conditions, with or without bFGF and heparin, primary corneal fibroblasts appeared morphologically similar to in situ corneal keratocytes, demonstrating a broad, stellate morphology with interconnected processes and no α-SM actin expression. Addition of TGF(β1) to serum-free cultures resulted in a dramatic transformation of corneal keratocytes to spindle-shaped, fibroblast-like cells that expressed α-SM actin in 100% of cells and exhibited a 20-fold increase in fibronectin synthesis and a 13-fold increase in α5β1-integrin synthesis. These effects were blocked by the addition of neutralizing antibodies (16 μ/ml). Overall these data suggest that TGF(β1) is a potent modulator of myofibroblast transformation under serum- free conditions. In addition, the growth of keratocytes in serum appears to mimic, in part, in vivo activation and myofibroblast transformation. We conclude that detailed study of TGF(β1)-induced myofibroblast transformation under defined serum-free conditions will provide important insights into the myofibroblast transformation process.

Original languageEnglish (US)
Pages (from-to)505-516
Number of pages12
JournalCornea
Volume15
Issue number5
StatePublished - 1996

Fingerprint

Corneal Keratocytes
Myofibroblasts
Smooth Muscle
Actins
Transforming Growth Factors
Serum
Fibroblast Growth Factor 2
Heparin
Fibroblasts
Fibronectins
Integrins
Fibroblast Growth Factor 10
Integrin alpha5beta1
Personal Autonomy
Neutralizing Antibodies
Immunoprecipitation
Fluorescent Antibody Technique

Keywords

  • α-Actin
  • αβ-Integrin
  • Cornea
  • Fibroblasts
  • Myofibroblasts

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Induction of α-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes. / Jester, James V.; Barry-Lane, Patricia A.; Cavanagh, Harrison D; Petroll, Walter M.

In: Cornea, Vol. 15, No. 5, 1996, p. 505-516.

Research output: Contribution to journalArticle

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abstract = "The effects of serum, transforming growth factor (TGF)(β1), bFGF, and heparin on in vitro myofibroblast transformation was studied. Primary rabbit corneal keratocytes were grown under serum-free conditions or in media supplemented with serum (10{\%} fetal calf serum), TGF(β1) (0.1-10 ng/ml), basic fibroblast growth factor (bFGF) (0.1-10 ng/ml), or heparin (10 U/ml). Cells were analyzed for expression of α-smooth muscle actin (α-SM actin), α5β1 integrin (the high-affinity fibronectin receptor) and fibronectin by immunoprecipitation, Western blotting, and immunofluorescence. Corneal keratocytes grown in the presence of serum showed a typical fibroblast morphology with induction of α-SM actin expression in 1 to 10{\%} of cells. Addition of bFGF blocked serum-induced α-SM actin expression, whereas addition of TGF(β1) enhanced α-SM actin expression (100{\%}), which in combination with heparin (10 U/ml), led to a pulling apart of the fibroblastic sheet, simulating contraction. Under serum-free conditions, with or without bFGF and heparin, primary corneal fibroblasts appeared morphologically similar to in situ corneal keratocytes, demonstrating a broad, stellate morphology with interconnected processes and no α-SM actin expression. Addition of TGF(β1) to serum-free cultures resulted in a dramatic transformation of corneal keratocytes to spindle-shaped, fibroblast-like cells that expressed α-SM actin in 100{\%} of cells and exhibited a 20-fold increase in fibronectin synthesis and a 13-fold increase in α5β1-integrin synthesis. These effects were blocked by the addition of neutralizing antibodies (16 μ/ml). Overall these data suggest that TGF(β1) is a potent modulator of myofibroblast transformation under serum- free conditions. In addition, the growth of keratocytes in serum appears to mimic, in part, in vivo activation and myofibroblast transformation. We conclude that detailed study of TGF(β1)-induced myofibroblast transformation under defined serum-free conditions will provide important insights into the myofibroblast transformation process.",
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