Abstract
Human recombinant basic fibroblast growth factor (bFGF) has been available for the treatment for non-healing skin ulcer, however, there still exist unknown biological effects on wound healing. Fibroblast-collagen matrix has been used as a model system to study how cells organize connective tissue. Previous our works showed that PI3K, Rac and Rho kinase are involved in bFGF-stimulated collagen matrix contraction. Recent clinical reports have pointed out bFGF promotes scarless wound healing. The mechanisms, however, are still unclear. The current studies were carried out to elucidate the mechanisms. It has been reported that transforming growth factor-β1 (TGF-β1) was required to activate fibroblasts to express the myofibroblast phenotype in vitro, i.e., increased expression of α-smooth muscle actin (αSMA). In the present study, we employed this technique to obtain myofibroblasts.
Levels of cellular αSMA increased after 2-4 days of TGF-β1 treatment alone and were consistently elevated after 5 days. However, levels of cellular αSMA increased 4-6 days after costimulation of bFGF and TGF-β1. Although spontaneous contraction was seen in stressed myofibroblast-collagen matrix, bFGF could cancel the gel contraction. There were not significant differences on cell spreading in myofibroblast-collagen gel, however, the number of cells was decreased when they were stimulated with bFGF. bFGF stimulation for myofibroblasts as well as fibroblasts caused transient Rac and Rho activation. Levels of diphosphorylated myosin light chain (MLC) were highest in fibroblasts 30 mins after bFGF stimulation. In contrast, basal level of diphosphorylated MLC was elevated in myofibroblasts without bFGF stimulation. After bFGF stimulation, levels of diphosphorylated MLC were further elevated up to 60 mins. bFGF promoted apoptosis in myofibroblasts but not in fibroblasts even if two different inhibitors, LY294002 for phosphatidylinositol-3-Kinase and Akt inhibitor, were present.
The present study implicates that the down regulation of PI3K to Akt pathway is involved in bFGF-promoted myofibroblast apoptosis. These results suggest that bFGF can promote scarless wound healing consequent upon induction of apoptosis in myofibroblasts.
