Release of FGF1 and Pathology of Angiogenesis

性状（由申请方提供）：FGF 1是一种强效促血管生成生长因子。重组FGF 1已在体内用于刺激血管生长以响应心血管损伤。开发有效的方法来调节内源性FGF 1的可用性可以有助于治疗各种心血管疾病。与大多数分泌蛋白不同，FGF 1缺乏经典的信号肽。因此，为了实现其生物学功能，FGF 1通过应激诱导的ER-高尔基体非依赖性途径释放。FGF 1作为铜依赖性多蛋白释放复合物（MRC）输出。FGF 1释放通过大的细胞膜结构域进行，其特征在于膜起泡和磷脂酰丝氨酸的外化。值得注意的是，与FGF 1共享几乎相同的3D结构的无信号肽促炎细胞因子白细胞介素11表现出类似的应激诱导释放机制。铜螯合物在体外抑制FGF 1和IL 11的释放，抑制大鼠动脉再狭窄和大鼠佐剂性关节炎的发生。该竞争性更新申请的目的是阐明FGF 1 MRC组装和释放的机制，并评估FGF 1释放对血管系统的影响。我们提出以下假设：在应激期间，FGF 1 MRC组分被转运至质膜的特定结构域并与之相互作用，在质膜中它们经历铜依赖性组装和进一步易位，所述易位由它们的膜去稳定特性和酸性磷脂的跨膜翻转限定。提出了以下具体目标：1。表征FGF 1 MRC组分与质膜的相互作用。2.了解FGF 1 MRC的组装及其通过出口门的释放。3.建立可诱导细胞类型特异性表达FGF 1的转基因小鼠，并利用它们探索FGF 1在体内的输出及其血管效应。特异性目的1和2将使用共聚焦免疫荧光显微镜（包括FRET和FICS方法）、细胞膜分级分离、蛋白质诱变、脂质体和细胞膜囊泡研究、膜下肌动蛋白细胞骨架特异性调节剂的表达。在第三部分中，我们将利用在内皮细胞和巨噬细胞/单核细胞中诱导表达FGF 1的转基因小鼠，研究FGF 1在机体内的输出调节及其对缺血肢体再狭窄和血管再生的影响。FGF 1是一种强有力的促血管生成生长因子，参与心血管系统的发育、维持和对损伤的反应。FGF 1通过特定的应激诱导的非经典释放途径递送至靶细胞。该提案的目标是了解FGF 1输出的机制以及该过程在心血管病理学中的作用。

DESCRIPTION (provided by applicant): FGF1 is a potent pro-angiogenic growth factor. Recombinant FGF1 has been used in vivo to stimulate vessel growth in response to cardiovascular damage. Development of efficient approaches to the regulation of the availability of endogenous FGF1 can contribute to the treatment of a wide range of cardiovascular disorders. Unlike most secreted proteins, FGF1 lacks a classical signal peptide. Thus, to realize its biological functions, FGF1 is released through the stress-induced ER-Golgi-independent pathway. FGF1 is exported as a copper-dependent multiprotein release complex (MRC). FGF1 release proceeds through large cell membrane domains characterized by membrane blebbing and externalization of phosphatidylserine. Significantly, the signal peptide-less pro-inflammatory cytokine interleukin 11, which shares an almost identical 3D structure with FGF1, exhibits a similar mechanism of stress-induced release. Copper chelation known to repress the release of FGF1 and IL11 in vitro, inhibits the restenosis in rat arteries and development of adjuvant induced arthritis in rats. The goal of this competitive renewal application is to elucidate the mechanisms of FGF1 MRC assembly and release and to assess the effect of FGF1 release upon the vasculature. We propose the hypothesis that during stress, FGF1 MRC components are transported to and interact with specific domains of the plasma membrane where they undergo copper- dependent assembly and further translocation defined by their membrane destabilizing properties and transmembrane flipping of acidic phospholipids. The following Specific Aims are proposed: 1. Characterize the interaction of FGF1 MRC components with the plasma membrane. 2. Understand the assembly of the FGF1 MRC and its release through the exit gates. 3. Develop transgenic mice with inducible cell type-specific FGF1 expression and use them to explore FGF1 export in vivo and its vascular effects. Confocal immunofluorescence microscopy (including FRET and FICS methods), cell membrane fractionation, protein mutagenesis, studies on liposomes and cell membrane vesicles, expression of specific regulators of the submembrane actin cytoskeleton will be used in Specific Aims 1 and 2. In the Specific Aim 3, we will use transgenic mice with the inducible expression of FGF1 in endothelial cells and macrophages/monocytes to study the regulation of FGF1 export in the organism and its effect upon restenosis and restorative angiogenesis in ischemic limbs.FGF1 is a potent pro-angiogenic growth factor which participates in the development of cardiovascular system, its maintenance and response to damage. FGF1 is delivered to target cells through a specific stress-induced non-classical release pathway. The goal of this proposal is to understand the mechanism of FGF1 export and the role of this process in cardiovascular pathologies.