Engineering FGF-1 for Increased Angiogenicity

描述(申请人提供)：严重的肢体缺血每年导致150,000例非创伤性的下肢截肢，其中一半以上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，可以用来增加糖尿病患者缺血肢体的血流量。这一建议是基于这样一个假设，即成纤维细胞生长因子-1(成纤维细胞生长因子-1)可以通过构建具有血管靶向序列的嵌合体并使用定点突变来修改参与凝血酶降解和肝素依赖的特定氨基酸序列来增加血管生成能力。 将合成一种与血管生成内皮细胞(NGR)结合的肽序列融合的成纤维细胞生长因子-1嵌合体，并对其进行鉴定。这种嵌合体(NGR/成纤维细胞生长因子-1)对内皮、平滑肌和壁前体细胞的体外有丝分裂活性将被确定，以及它对凝血酶降解和肝素依赖的敏感性。纤维蛋白凝胶模型将用于量化NGR/成纤维细胞生长因子-1的体外血管生成能力，并确定其与血管萌芽相关的3D空间定位。 然后，将使用新的3D技术和简单的胶原胶模型来量化NGR/FGF-1和其他先前设计的突变体的体内血管生成能力。这种植入糖尿病小鼠模型的凝胶将被用来识别血管生成能力最强的生长因子。然后，将评估这两种最具血管生成能力的生长因子在糖尿病小鼠严重肢体缺血模型中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.