Construction and biological characterization of an HB-GAM/FGF-1 chimera for vascular tissue engineering

Objective: Cardiovascular tissue engineering approaches to vessel wall restoration have focused on the potent but relatively nonspecific and heparin-dependent mesenchymal cell mitogen fibroblast growth factor 1 (FGF-1). We hypothesized that linking FGF-1 to a sequence likely to bind to cell surface receptors relatively more abundant on endothelial cells (ECs) might induce a relative greater EC bioavailability of the FGF-1. We constructed a heparin-binding growth-associated molecule (HB-GAM)/FGF-1 chimera by linking full-length human HB-GAM to the amino-terminus of human FGF-1β (21-154) and tested its activities on smooth muscle cells (SMCs) and ECs. Methods: Primary canine carotid SMCs and jugular vein ECs were plated in 96-well plates in media containing 10% fetal bovine serum and grown to approximately 80% confluence. After being growth arrested in serum-free media for 24 hours, the cells were exposed to concentration ranges of cytokines and heparin, and proliferation was measured with tritiated-thymidine incorporation. Twenty percent fetal bovine serum was used as positive control, and phosphate-buffered saline was used as negative control. Results: In the presence of heparin the HB-GAM/FGF-1 chimera stimulated less SMC proliferation than did the wild-type FGF-1 with a median effective dose of approximately 0.3 nmol versus approximately 0.1 nmol (P < .001). By contrast, the chimera retained full stimulating activity on EC proliferation with a median effective dose of 0.06 nmol for both cytokines. Unlike the wild-type protein, the chimera possessed heparin-independent activity. In the absence of heparin, the chimera induced dose-dependent EC and SMC proliferation at 0.06 nmol or more compared with the wild-type FGF-1, which stimulated minimal DNA synthesis at 6.0-nmol concentrations. Conclusions: The HB-GAM/FGF-1 chimera displays significantly greater and uniquely heparin-independent mitogenic activity for both cell types, and in the presence of heparin it displays a significantly greater EC specificity.