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 language||English (US)|
|Number of pages||12|
|State||Published - Aug 27 1996|
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