Myofibroblast transformation of cat corneal endothelium by transforming growth factor-beta1, -beta2, and -beta3.
OBJECTIVE
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)-beta 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-gamma1, TGF-beta2, TGF-beta3, basic fibroblast growth factor (bFGF), or TGF-beta1 and bFGF. Buttons were single and double labeled using phalloidin and antibodies to ZO-1, Ki67, fibronectin, alpha-smooth muscle (SM) actin, and vinculin. Counts of Ki67-positive cells were used as a measure of endothelial proliferation.


RESULTS
Organ culture in TGF-beta1, beta2, or beta3 induced myofibroblast transformation of corneal endothelial cells, with formation of stress fibers containing alpha-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-beta3 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-beta1 and bFGF resulted in both myofibroblast transformation and increased proliferation.


CONCLUSIONS
These results suggest that TGF-beta 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.
Petroll
W M
WM
Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, 75235-9057, USA.
Jester
J V
JV
Bean
J J
JJ
Cavanagh
H D
HD
D000818:Animals; D002415:Cats; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009924:Organ Culture Techniques; D010590:Phalloidine; D010641:Phenotype; D010750:Phosphoproteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D062826:Zonula Occludens-1 Protein
D016326:Extracellular Matrix Proteins; D019394:Ki-67 Antigen; D008565:Membrane Proteins; D010750:Phosphoproteins; D016212:Transforming Growth Factor beta; D062826:Zonula Occludens-1 Protein; D010590:Phalloidine