title: Induction of alpha-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes.
abstract: The effects of serum, transforming growth factor (TGF) beta 1, 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 beta 1 (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 alpha-smooth muscle actin (alpha-SM actin), alpha 5 beta 1 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 alpha-SM actin expression in 1 to 10% of cells. Addition of bFGF blocked serum-induced alpha-SM actin expression, whereas addition of TGF beta 1 enhanced alpha-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 alpha-SM actin expression. Addition of TGF beta 1 to serum-free cultures resulted in a dramatic transformation of corneal keratocytes to spindle-shaped, fibroblast-like cells that expressed alpha-SM actin in 100% of cells and exhibited a 20-fold increase in fibronectin synthesis and a 13-fold increase in alpha 5 beta 1-integrin synthesis. These effects were blocked by the addition of neutralizing antibodies (16 micrograms/ml). Overall these data suggest that TGF beta 1 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 beta 1-induced myofibroblast transformation under defined serum-free conditions will provide important insights into the myofibroblast transformation process.
authors.affiliation: Department of Ophthalmology, University of Texas, Southwestern Medical Center at Dallas 75235-9057, USA.
authors.forename: J V
authors.initials: JV
authors.lastname: Jester
authors.forename: P A
authors.initials: PA
authors.lastname: Barry-Lane
authors.forename: H D
authors.initials: HD
authors.lastname: Cavanagh
authors.forename: W M
authors.initials: WM
authors.lastname: Petroll
mesh_terms: D000199:Actins / Q000096:biosynthesis*; D000818:Animals; D015153:Blotting, Western; D002455:Cell Division; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005353:Fibronectins / Q000096:biosynthesis; D019085:Fluorescent Antibody Technique, Direct; D019084:Fluorescent Antibody Technique, Indirect; D006493:Heparin / Q000494:pharmacology; D009130:Muscle, Smooth / Q000378:metabolism*; D011233:Precipitin Tests; D011817:Rabbits; D017469:Receptors, Fibronectin / Q000096:biosynthesis; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D000199:Actins; D005353:Fibronectins; D017469:Receptors, Fibronectin; D016212:Transforming Growth Factor beta; D016222:Fibroblast Growth Factor 2; D006493:Heparin