Engineering FGF-1 for Increased Angiogenicity

描述（由申请人提供）：严重肢体缺血导致每年150，000例非创伤性下肢截肢，其中超过一半发生在糖尿病患者中。这项研究的长期目标是设计一种生长因子，可用于增加糖尿病患者缺血肢体的血流量。该提议是由以下假设驱动的：成纤维细胞生长因子-1（FGF-1）可以通过创建具有血管靶向序列的嵌合体并使用定点诱变来修饰参与凝血酶降解和肝素依赖性的特定氨基酸序列而被工程化以增加血管生成。 将合成并表征与结合血管生成内皮细胞（NGR）的肽序列融合的FGF-1嵌合体。将测定该嵌合体（NGR/FGF-1）对内皮细胞、平滑肌细胞和壁前体细胞的体外促有丝分裂性，以及其对凝血酶降解和肝素依赖性的敏感性。纤维蛋白凝胶模型将用于量化NGR/FGF-1的体外血管生成性，并确定其与血管芽相关的3D空间定位。 NGR/FGF-1和其他先前设计的FGF-1突变体的体内血管生成性将使用新的3D技术和简单的胶原凝胶模型进行定量。这种凝胶植入糖尿病小鼠模型中，将用于鉴定具有最强血管生成性的生长因子。然后，在严重肢体缺血的糖尿病小鼠模型中评估两种最具血管生成性的生长因子促进血管生成和增加血流量的能力。

DESCRIPTION (provided by applicant): 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.