The effect of factor VII activating protease treatment on proliferation and collagen synthesis of cultured human pulmonary fibroblasts

Abstract

To examine the influence of factor VII-activating protease (FSAP) treatment on cell proliferation and collagen synthesis in human pulmonary fibroblasts (HPF). Cultured HPF were treated with heparin (10 mg/L), platelet-derived growth factor-BB (PDGF-BB, 20 μg/L), FSAP (12 mg/L), aprotinin (10 mg/L) separately and in different mixture. The proliferation of the HPF was determined by 5-bromodeoxyuridine (BrDU) incorporation. The collagen III mRNA was determined by real-time polymerase chain reaction (PCR), and its protein expression, as well as the phosphorylation of p42 / p44 mitogen activated protein kinase (MAPK) were both determined using Western blotting. PDGF BB significantly (all P < 0.05) induced cell proliferation (1.23 ± 0.06 vs. 1.01 ± 0.01 without heparin, 1.24 ± 0.04 vs. 0.98 ± 0.01 with heparin), collagen III mRNA transcription (1.79 ± 0.02 vs. 1.00 ± 0.00 without aprotinin, 1.57 ± 0.01 vs. 1.00 ± 0.00 with aprotinin), and collagen III protein expression (0.54 ± 0.26 vs. 0.17 ± 0.05 without aprotinin, 0.59 ± 0.31 vs. 0.24 ± 0.15 with aprotinin) in HPF. PDGF BB also significantly (both P < 0.05) induced p42 / p44 MAPK phosphorylation in HPF (0.89 ± 0.24 vs. 0.51 ± 0.17 without aprotinin, 0.97 ± 0.41 vs. 0.37 ± 0.05 with aprotinin). In the presence of heparin FSAP significantly (all P < 0.05) inhibited the PDGF BB induced HPF proliferation (0.92 ± 0.23 vs 1.19 ± 0.11); collagen III mRNA transcription (0.61 ± 0.02 vs. 1.79 ± 0.02) and collagen III protein expression (0.15 ± 0.12 vs. 0.54 ± 0.26). FSAP also inhibited PDGF induced p42 / p44 MAPK phosphorylation in HPF (0.57 ± 0.16 vs. 0.89 ± 0.24) significantly (P < 0.05). The presence of aprotinin lead to a reversal of the inhibitory effect of FSAP on collagen III mRNA transcription (2.37 ± 0.21 vs.0.61 ± 0.02), collagen III protein expression (0.55 ± 0.24 vs. 0.15 ± 0.12) and p42 / p44 MAPK phosphorylation (0.86 ± 0.41 vs. 0.57 ± 0.16, all P < 0.05). FSAP could inhibit the PDGF-BB induced proliferation and collagen III synthesis in HPF in vitro through the suppression of p42 / p44 MAPK phosphorylation.