Critical limb ischemia results in 150,000 non-traumatic lower limb amputations annually, with over half occurring in diabetic patients. The long-term goal of this research is to design a growth factor that can be used to increase blood flow in ischemic limbs of diabetic patients. This proposal is driven by the hypothesis that fibroblast growth factor-one (FGF-1) can be engineered for increased angiogenicity by creating chimeras with vascular targeting sequences and using site directed mutagenesis to modify specific amino acids sequences involved in thrombin degradation and heparin dependence. A chimera of FGF-1 fused with a peptide sequence that binds angiogenic endothelial cells (NGR) will be synthesized and characterized. The in vitro mitogenicity of this chimera (NGR/FGF-1) for endothelial, smooth muscle, and mural precursor cells will be determined, as will its susceptibility to thrombin degradation and heparin dependence. A fibrin gel model will be used to quantify the in vitro angiogenicity of NGR/FGF-1 and determine its 3D spatial localization in relation to vessel sprouts. The in vivo angiogenicity of NGR/FGF-1 and other previously designed FGF-1 mutants will then be quantified using a novel 3D technique and a simple collagen gel model. This gel, implanted in a mouse model of diabetes will be used to identify the growth factors with the strongest angiogenicity. The two most angiogenic growth factors will then be assessed for their ability to promote angiogenesis and increase blood flow in a diabetic mouse model of critical limb ischemia.
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