Release of FGF1 and Pathology of Angiogenesis

描述(申请人提供)：FGF1是一种有效的促血管生成生长因子。重组FGF1已在体内用于刺激血管生长以应对心血管损伤。开发有效的方法来调节内源性FGF1的可获得性，有助于治疗广泛的心血管疾病。与大多数分泌蛋白不同，FGF1缺乏经典的信号肽。因此，为了实现其生物学功能，FGF1是通过应激诱导的ER-高尔基体非依赖性途径释放的。FGF1以铜依赖的多蛋白释放复合体(MRC)的形式输出。FGF1的释放通过大的细胞膜域进行，其特征是膜起泡和磷脂酰丝氨酸的外化。值得注意的是，与FGF1具有几乎相同的3D结构的信号肽非致炎细胞因子白介素11表现出类似的应激诱导释放机制。铜络合在体外可抑制FGF1和IL11的释放，抑制大鼠动脉再狭窄和大鼠佐剂性关节炎的发展。这一竞争性更新应用的目的是阐明FGF1 MRC组装和释放的机制，并评估FGF1释放对血管系统的影响。我们提出的假设是，在胁迫过程中，FGF1 MRC组分被运输到质膜的特定区域并与其相互作用，在那里它们经历了铜依赖的组装和进一步的易位，这取决于它们的膜不稳定特性和酸性磷脂的跨膜翻转。具体目标如下：1.表征FGF1 MRC组分与质膜的相互作用。2.了解FGF1 MRC的组装和通过出口门的释放。3.建立可诱导细胞类型特异性FGF1表达的转基因小鼠，并用于研究FGF1在体内的输出及其血管效应。共聚焦免疫荧光显微镜(包括FRET和FICS方法)、细胞膜分离、蛋白质突变、脂质体和细胞膜囊泡的研究、膜下肌动蛋白细胞骨架特异性调节因子的表达将用于特定的目标1和2。在特定的目标3中，我们将使用在内皮细胞和巨噬细胞/单核细胞中诱导表达FGF1的转基因小鼠来研究FGF1在生物体内的输出调节及其对缺血肢体再狭窄和恢复性血管生成的影响。FGF1是一种强大的促血管生成生长因子，参与心血管系统的发育及其对损伤的维持和反应。FGF1通过特定的应激诱导的非经典释放途径被传递到靶细胞。这项建议的目的是了解FGF1输出的机制以及这一过程在心血管病理中的作用。

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.