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Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.
Fibroblast growth factor reversal of the corneal myofibroblast phenotype.
Investigative Ophthalmology & Visual Science 2001 October
PURPOSE: Keratocytes give rise to fibroblasts and myofibroblasts in wounded cornea. It is well established that treatment of fibroblasts with transforming growth factor (TGF) beta will induce myofibroblast differentiation. We investigated whether this differentiation could be reversed by the administration of fibroblast growth factor (FGF).
METHODS: Cultured corneal myofibroblasts were plated at 200 cells/mm(2), and cells were grown in DMEM/F12 containing (1) 10% FBS or (2) 10% FBS with FGF and heparin or (3) 1% FBS or (4) 1% FBS with TGF-beta. As distinguished from the fibroblast phenotype, the myofibroblast phenotype was identified by the assembly of alpha-smooth muscle (SM) actin protein into the stress fiber cytoskeleton. To further characterize growth factor regulation of the two phenotypes, the phenotypic expression of TGF-beta receptor types I and II, cadherins, and connexin 43 by immunocytochemistry, Western blot analysis, and immunoprecipitation and of alpha-SM actin mRNA in Northern blot analysis were evaluated.
RESULTS: Corneal myofibroblasts replated and grown in the presence of FGF-1 or FGF-2 (20 ng/ml) plus heparin (5 microg/ml) in 10% FBS medium had decreased expression of alpha-SM actin protein, TGF-beta receptors, and cadherins. Thus, FGF-heparin decreased the myofibroblast phenotype and promoted the fibroblast phenotype. Administration of either 20 ng/ml FGF or 5 microg/ml heparin alone was not effective. Addition of TGF-beta further enhanced the expression of alpha-SM actin mRNA and protein and cell surface expression of TGF-beta receptors in myofibroblast cultures.
CONCLUSIONS: FGF-1 or -2 and heparin promoted the fibroblast phenotype and reversed the myofibroblast phenotype. This finding supports the idea that corneal myofibroblasts and fibroblasts are alternative phenotypes rather than terminally differentiated cell types.
METHODS: Cultured corneal myofibroblasts were plated at 200 cells/mm(2), and cells were grown in DMEM/F12 containing (1) 10% FBS or (2) 10% FBS with FGF and heparin or (3) 1% FBS or (4) 1% FBS with TGF-beta. As distinguished from the fibroblast phenotype, the myofibroblast phenotype was identified by the assembly of alpha-smooth muscle (SM) actin protein into the stress fiber cytoskeleton. To further characterize growth factor regulation of the two phenotypes, the phenotypic expression of TGF-beta receptor types I and II, cadherins, and connexin 43 by immunocytochemistry, Western blot analysis, and immunoprecipitation and of alpha-SM actin mRNA in Northern blot analysis were evaluated.
RESULTS: Corneal myofibroblasts replated and grown in the presence of FGF-1 or FGF-2 (20 ng/ml) plus heparin (5 microg/ml) in 10% FBS medium had decreased expression of alpha-SM actin protein, TGF-beta receptors, and cadherins. Thus, FGF-heparin decreased the myofibroblast phenotype and promoted the fibroblast phenotype. Administration of either 20 ng/ml FGF or 5 microg/ml heparin alone was not effective. Addition of TGF-beta further enhanced the expression of alpha-SM actin mRNA and protein and cell surface expression of TGF-beta receptors in myofibroblast cultures.
CONCLUSIONS: FGF-1 or -2 and heparin promoted the fibroblast phenotype and reversed the myofibroblast phenotype. This finding supports the idea that corneal myofibroblasts and fibroblasts are alternative phenotypes rather than terminally differentiated cell types.
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