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.
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