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

Objective: Cardiovascular tissue engineering approaches to vessel wall rest oration have focused on the potent but relatively nonspecific and heparin-d ependent mesenchymal cell mitogen fibroblast growth factor 1 (FGF-1). We hy pothesized 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 hepari n-binding growth-associated molecule (HB-GAM)FGF-1 chimera by linking full- length human HB-GAM to the amino-terminus of human FGF-1 beta (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 approx imately 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 incorporat ion. Twenty percent fetal bovine serum was used as positive control, and ph osphate-buffered saline was used as negative control. Results: In the presence of heparin the HB-GAM/FGF-1 chimera stimulated les s SMC proliferation than did the wild-type FGF-1 with a median effective do se of approximately 0.3 nmol versus approximately 0.1 nmol (P <.001). By co ntrast, the chimera retained full stimulating activity on EC proliferation with a median effective dose of 0.06 nmol for both cytokines. Unlike the wi ld-type protein, the chimera possessed heparin-independent activity: In the absence of heparin, the chimera induced dose-dependent EC and SMC prolifer ation at 0.06 nmol or more compared with the wild-type FGF-1, which stimula ted minimal DNA synthesis at 6.0-nmol concentrations. Conclusions: The HB-GAM/FGF-1 chimera displays significantly greater and un iquely heparin-independent mitogenic activity for both cell types, and in t he presence of heparin it displays a significantly greater EC specificity.