Simultaneous application of basic fibroblast growth factor and hcpatocyte growth factor to enhance the blood vessels formation

Objective: The present study investigated whether the simultaneous application of basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) enhances blood vessel formation in murine ischemic hindlimb compared with bFGF or HGF applied alone. Methods. Unilateral hindlimb ischemia was created in C57BL/6 mice. Hindlimb blood flow was evaluated by laser Doppler perfusion image index (LDPII) (ratio (%) of ischemic-to-normal-limb blood flow). The ischemic limbs were treated with bFGF and HGF separately, or bFGF and HGF together, and their therapeutic effects were assessed. Collagen microspheres (CM) were used as a sustained-release carrier for bFGF and HGF. Results. A single intramuscular injection of 5 mug or less of bFGF-incorporated CM (bFGF/CM) into the ischemic limb did not significantly increase the LDPII compared with the control (no treatment) 4 weeks after the treatment. Similarly, 20 mug or less of HGF/CM did not increase LDPII Based on these results, we compared the dual release of CM incorporating 5 mug of bFGF and 20 mug of HGF with either the single release of 5 mug of bFGF/CM alone or 20 mug of HGF/CM alone. The LDPII of the dual release (94.2% +/- 10.9%) was higher than either single release (51.2% +/- 5.8% or 52.5% +/- 8.0%, P <.01). Furthermore, the LDPII in the dual release (94.2% +/- 10.9%) was equivalent to that with 80 mug of bFGF/CM (95.1% +/- 7.6%) alone or 80 mug of HGF/CM (92.8% +/- 7.6%) alone. A histologic evaluation at 4 weeks showed capillary density in the dual release (868 +/- 173 vessels/mm(2)) was higher than that in either single release (204 +/- 68 vessels/mm(2) or 185 98 vessels/mm(2), p <.01). The percentage of mature vessels assessed by a-smooth muscle actin staining was also higher in the dual release (43.8% +/- 7.8% vs 9.5% +/- 3.0% or 11.7% +/- 3.8%, respectively; P <.01). Conclusions: This study demonstrates that the sustained dual release of a lower dose of bFGF and HGF from a carrier matrix can achieve equivalent blood perfusion recovery and more mature vasculature in the ischemic limb than a higher dose of bFGF or HGF alone. This approach may be a highly promising strategy for the future treatment of peripheral vascular disease. (J Vase Surg 2005;41:82-90.) Clinical Relevance: Clinical trials of growth-factor therapies for ischemia have revealed several problems, such as immature vasculature, adverse effects of high-dose growth factors, or immune and inflammatory responses of viral vectors. This study demonstrates that the sustained dual release of lower-dose basic fibroblast growth factor (bFGF) and hepatocyte growth factor (FIGF) from biodegradable collagen microspheres (CM) can achieve equivalent blood perfusion recovery and more mature vasculature in ischemic limb than higher-dose bFGF or HGF alone. CM does not aggravate focal inflammation in the ischernic limb. This approach may be a highly promising strategy for the future treatment of peripheral vascular disease.