INDUCTION OF ALPHA-SMOOTH MUSCLE ACTIN EXPRESSION AND MYOFIBROBLAST TRANSFORMATION IN CULTURED CORNEAL KERATOCYTES

The effects of serum, transforming growth factor (TGF)(beta 1), bFGF, and heparin on in vitro myofibroblast transformation was studied. Prim ary rabbit corneal keratocytes were grown under serum-free conditions or in media supplemented with serum (10% fetal calf serum), TGF (0.1-1 0 ng/ml), basic fibroblast growth factor (TGF(beta 1)) (0.1-10 ng/ml), or heparin (10 U/ml). Cells were analyzed for expression of alpha-smo oth 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 i nduction of alpha-SM actin expression in 1 to 10% of cells. Addition o f bFGF blocked serum-induced a-SM actin expression, whereas addition o f beta(1) enhanced alpha-SM actin expression (100%), which in TGF comb ination with heparin (10 U/ml), led to a pulling apart of the fibrobla stic sheet, simulating contraction. Under serum-free conditions, with or without bFGF and heparin, primary corneal fibroblasts appeared morp hologically similar to in situ corneal keratocytes, demonstrating a br oad, stellate morphology with interconnected processes and no alpha-SM actin expression. Addition of TGF(beta 1) to serum-free cultures resu lted in a dramatic transformation of corneal keratocytes to spindle-sh aped, fibroblastlike cells that expressed alpha-SM actin in 100% of ce lls and exhibited a 20-fold increase in fibronectin synthesis and a 13 -fold increase in alpha(5) beta(1)-integrin synthesis. These effects w ere blocked by the addition of neutralizing antibodies (16 mu g/ml). O verall these data suggest that TGF beta, is a potent modulator of myof ibroblast transformation under serum-free conditions. In addition, the growth of keratocytes in serum appears to mimic, in part, in vivo act ivation and myofibroblast transformation. We conclude that detailed st udy of TGF(beta 1)-induced myofibroblast transformation under defined serum-free conditions will provide important insights into the myofibr oblast transformation process.