Acidic fibroblast growth factor decreases alpha-smooth muscle actin expression and induces apoptosis in human normal lung fibroblasts

Abstract

Fibroblast/myofibroblast expansion is critical in the pathogenesis of pulmonary fibrosis. To date, most research has focused on profibrotic mediators, whereas studies on antifibrotic factors are scanty. In this study, we explored the effects of acidic fibroblast growth factor (FGF-1) and FGF-1 plus heparin (FGF-1+H) on fibroblast growth rate, apoptosis, and myofibroblast differentiation. Heparin was used because it participates in FGF-1 signaling. Growth rate was evaluated by WST-1 colorimetric assay, DNA synthesis by [H-3]thymidine incorporation, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cleaved caspase 3. Expression of alpha-smooth muscle actin (alpha-SMA) was examined by immunocytochemistry, flow cytometry, real-time PCR, and immunoblotting. Despite the induction of DNA synthesis, FGF-1+H significantly reduced fibroblast growth rate. This correlated with a significant increase in apoptosis, evaluated by TUNEL (41.6 +/- 1.4% vs. 12.5 +/- 0.6% from controls

P < 0.01) and cleaved caspase 3 (295 +/- 32 vs. 200 +/- 19 ng/10(6) cells from controls

P < 0.05). Double immunostaining (alpha-SMA-TUNEL) revealed that the levels of induced apoptosis were similar in fibroblasts and myofibroblasts. FGF-1+H inhibited the effect of TGF-beta 1 on myofibroblast differentiation. alpha-SMA-positive cells were reduced by immunocytochemistry from 44.5 +/- 6.5% to 10.9 +/- 1.9% and by flow cytometry from 30.6 +/- 2.5% to 7.7 +/- 0.6% (P < 0.01). Also, FGF-1+H significantly inhibited the TGF-beta 1 induction of alpha-SMA quantified by real-time PCR and Western blot. This decrease was associated with a 35% reduction in TGF-beta 1-induced collagen gel contraction. The effect of FGF-1+H was mediated by a significant decrease of TGF-beta 1-induced Smad2 phosphorylation. FGF-1 alone exhibited similar but lower effects. These findings suggest that FGF- 1 can have an antifibrogenic role, inducing apoptosis of fibroblasts and inhibiting myofibroblast differentiation.