Release of FGF1 and the Pathology of Angiogenesis

本实验室的长期目标是阐明成纤维细胞生长因子（FGF） 1在体外调节人内皮细胞迁移和增殖的机制，并将这一信息应用于体内组织和器官修复过程中增强血管生成反应。虽然最近FGF原型在应对缺血性心血管损伤的人类侧支血管生长中的应用证明了这一前提的有效性，但对FGF基因家族成员的结构-功能研究表明，我们对FGF功能的理解仍然是原始的。事实上，FGF原型中的两个新结构特征的功能尚不清楚，其中包括功能性核/核核定位信号的存在和缺乏经典的信号肽序列，通过传统的er -高尔基体介导途径指导其分泌。有趣的是，这些特征也存在于白细胞介素（IL）1基因家族成员中，该基因家族与FGF基因家族成员具有结构和晶体相似性。由于FGF1必须通过其高亲和力受体酪氨酸激酶进入细胞外隔室发出信号，以促进生物反应并进行受体依赖的核/核核易位，因此我们利用该奖项的资源来定义FGF1释放的机制。事实上，在目前的资助期内，我们已经证明FGF1而非FGF2以凋亡无关的方式释放，以响应生物应激，包括热休克、缺氧和血清剥夺。对应激诱导的FGF1释放途径的结构-功能分析表明，FGF1作为潜在的Cys30 FGF1同型二聚体被输出到细胞外室。FGF1同型二聚体是一种非共价高分子量复合物的组成部分，该复合物包括胞外运输蛋白p65 synaptotagmin （Syt1）的囊外结构域（p40）和膜联蛋白（Anx）2结合蛋白S100A13。有趣的是，像FGF1和IL1alpha一样，这些蛋白也缺乏经典的信号肽序列，并且每个蛋白都能够与酸性磷脂（包括磷脂酰丝氨酸）相关联。此外，我们提供的初步数据表明，负责释放无信号肽的促炎细胞因子IL1alpha的成熟而非前体形式的途径与FGF1使用的途径相似，并且IL1alpha的前体结构域在应激诱导的FGF1释放中起主要的负作用。因此，我们请求支持以确认和扩展这些结果，并建议通过(i)定义FGF1释放的结构先决条件和（ii）表征这些多肽促进FGF输出的细胞机制来进一步定义FGF1释放的机制。我们认为，这些研究可能不仅为血管生成的调控提供了新的见解，而且还可能为人类潜在的治疗管理提供新的药理学靶点。

The long-term goal of this laboratory has been to elucidate the mechanism by which fibroblast growth factor (FGF) 1 regulates the migration and proliferation of human endothelial cells in vitro and to apply this information to augment angiogenic responses during tissue and organ repair in vivo. While the recent application of the FGF prototypes for the generation of human collateral vessel growth in response to ischemic cardiovascular damage argues for the validity of this premise, structure-function studies with the members of the FGF gene family suggest that our understanding of FGF function is still primitive. Indeed, the function of two novel structural features within the FGF prototypes are not well understood and these include the presence of a functional nuclear/nucleolar localization signal(s) and the absence of a classical signal peptide sequence to direct its secretion through the conventional ER-Golgi apparatus-mediated pathway. Interestingly, these features are also found within members of the interleukin (IL)1 gene family which shares structural and crystallographic similarities with members of the FGF gene family. Since FGFs must gain access to the extracellular compartment to signal through their high affinity receptor tyrosine kinases in order to promote a biological response and undergo receptor-dependent nuclear/nucleolar translocation, we have utilized the resources from this award to define the mechanism by which FGF1 is released. Indeed, we have demonstrated during the current funding period that FGF1 but not FGF2 is released in response to biological stresses including heat shock, hypoxia, and serum deprivation in an apoptotic-independent manner. Structure- function analysis of the stress-induced FGF1 release pathway has demonstrated that FGF1 is exported into the extracellular compartment as a latent Cys30 FGF1 homodimer. The FGF1 homodimer is a component of a non-covalent high molecular weight complex which includes the extravesicular domain (p40) of the exocytotic trafficking-protein, p65 synaptotagmin (Syt1) and the annexin (Anx)2-binding protein, S100A13. Interestingly, like FGF1 and IL1alpha, these proteins also lack a classical signal peptide sequence and each is able to associate with acidic phospholipids including phosphatidylserine. In addition, we present preliminary data suggesting that the pathway responsible for the release of the mature but not the precursor form of the signal peptide-less pro-inflammatory cytokine, IL1alpha, is similar to the pathway utilized by FGF1 and that the precursor domain of IL1alpha acts as a dominant negative for the stress-induced release of FGF1. As a result, we request support to confirm and expand these results and propose to further define the mechanism of FGF1 release by (i) defining the structural prerequisites for the release of FGF1 and (ii) characterizing the cellular mechanisms by which these polypeptides facilitate FGF export. We suggest that these studies may not only provide new insight into the regulation of angiogenesis but may also enable access to novel pharmacologic targets for potential therapeutic management in man.