Background. The structure/function relationships of fribroblast growth factor 1 (FGF-1) are being investigated using site mutation, yielding novel structures with potential clinical applicability for modulating tissue responses to vascular interventions. We generated a mutant FGF-1 in which all three cysteines were convetted to serines and then tested the relative mitogenic activities on endothelial cells (ECs) and smooth muscle cells (SMCs) and the molecular stability of the protein to thrombin-induced degradation. Methods. The dose responses of wild-type FGF-1 and the Cys-free mutant in the absence or presence of heparin were tested on ECs and SMCs. Cell proliferation was measured by [3H]thymidine incorporation. Data were normalized by positive control (26% fetal bovine serum) and expressed as percentage of positive control for comparison. The molecular stability was examined by exposure of the cytokines to thrombin at 37°C for 0.5-24 l1 and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results. Unlike wild-type FGF-1 which induced only minimal DNA synthesis at concentrations as high as 100 ng/ml, the Cys-free mutant induced a dose- dependent proliferation starting at 1 ng/ml on both ECs and SMCs in the absence of heparin. At 100 ng/ml, Cys-free mutant induced 4-fold more proliferation than wild-type FGF-1 on ECs (76.64 ± 13.39% vs 14.58 ± 1.38%, P < 0.01) and 12-fold more proliferation on SMCs (143.52 ± 9.96 vs 11.25 ± 3.32, P < 0.01). Heparin 5 U/ml potentiated the mitogenic activity of the Cys-free mutant at low dose range. Both proteins were degraded by thrombin progressively. But the Cys-free mutant showed more susceptibility with accelerated appearance of lower-molecular-weight fragment bands after incubation with thrombin. Conclusions. Conversion of cysteine residues to serine changed the heparin dependency of the growth factor and increased its mitogenic activity and its susceptibility to thrombin-induced degradation. (C) 2000 Academic Press.
|Idioma original||English (US)|
|Número de artículo||95945|
|Número de páginas||6|
|Publicación||Journal of Surgical Research|
|Estado||Published - ago 2000|
|Publicado de forma externa||Sí|
ASJC Scopus subject areas