Myofibroblast transformation of cat corneal endothelium by transforming growth factor-β₁, -β₂, and -β₃

PURPOSE. Under certain pathophysiologic conditions, the corneal endothelium can produce an abnormal posterior collagenous layer (PCL) that reduces light transmission. Previous studies suggest that formation of PCLs can result from transformation of endothelial cells to a proliferative myofibroblast phenotype. The purpose of this study was to determine the potential role of transforming growth factor (TGF)-β on corneal endothelial transformation. METHODS. Three corneal buttons (6-mm diameter) were obtained from each cornea of 28 adult cats. After a 2-mm diameter mechanical scrape injury was made, each button was cultured for 24, 48, or 72 hours in serum- free medium (SFM) or SFM supplemented with 10% fetal calf serum, TGF-β₁, TGF-β₂, TGF-β₃, basic fibroblast growth factor (bFGF), or TGF-β₁ and bFGF. Buttons were single and double labeled using phalloidin and antibodies to ZO-1, Ki67, fibronectin, α-smooth muscle (SM) actin, and vinculin. Counts of Ki67-positive cells were used as a measure of endothelial proliferation. RESULTS. Organ culture in TGF-β₁, β₂, or β₃ induced myofibroblast transformation of corneal endothelial cells, with formation of stress fibers containing α-SM actin, loss of normal pericellular ZO-1 organization, development of extracellular fibronectin fibrils, and formation of focal contacts as indicated by punctate vinculin staining. However, TGF-β did not stimulate endothelial proliferation above that in serum-free control samples. Serum and bFGF each stimulated proliferation significantly, without inducing myofibroblast transformation. A combination of TGF-β₁ and bFGF resulted in both myofibroblast transformation and increased proliferation. CONCLUSIONS. These results suggest that TGF-β plays a key role in the loss of normal endothelial differentiation, abnormal extracellular matrix synthesis, and myofibroblast transformation, which can induce development of PCLs. However, other factors such as bFGF seem to be required to stimulate concomitant proliferation of corneal endothelium.