The investigators have studied the senescent phenotype of the human umbilical vein endothelial cell (HUVEC) in order to understand the mechanism of its non-replicative component. Prior studies from their laboratory have strongly implicated a prominent role for the cytokine, interleukin (IL)-1alpha as an inhibitor of fibroblast growth factor (FGF)-1-induced HUVEC and an inducer of HUVEC senescence in vitro. More recent studies have suggested that intracellular IL-1alpha act as an uncoupler of FGF receptor (R) signal transduction and indeed senescent HUVEC populations are not able to induce the tyrosine phosphorylation of the FGFR substrate Src even though exogenous FGF-1 is able to upregulate the steady-state mRNA levels of both immediate-early and mid-to-late FGF response genes. Because HUVEC populations are extremely difficult to stably transfect/transduce, it has been difficult to extend these observations and this recalcitrant behavior has limited progress. However, they have recently obtained stable precursor IL-1alpha transfectants using the ECV cell, a spontaneous HUVEC transformant, and data obtained from this system argues that precursor IL-1alpha but not the mature form of IL-1alpha is able to attenuate the proliferative and migratory potential of the ECV cell in vitro independent of the presence of exogenous IL-1 receptor antagonist (IRAP). These data demonstrate that precursor IL-1alpha functions as a intracellular repressor of ECV cell migration and growth. Interestingly, the precursor IL-1alpha but not the mature IL-1alpha ECV cell transfectants exhibit a prominent F-actin staining that strongly resembles the senescent HUVEC phenotype in vitro. Thus, to elucidate the mechanism of intracellular IL-1alpha precursor function the investigators propose to identify and characterize precursor IL-1alpha associated proteins in an attempt to elucidate the pathway of intracellular precursor IL-1alpha communication. They also propose to take advantage of the observation that the FGFR and IL-1R systems do not restrict the intracellular traffic or signaling capacities of the exogenous recombinant chimeric ligands, FGF-1:beta-galactosidase (gal) and precursor IL-1alpha:beta-gal. Thus, as an alternative to the inefficient transfection/transduction ability of HUVEC populations, they propose to construct and express a recombinant FGF-1:beta-gal:precursor IL-1alpha chimera. This dual ligand chimera should enable them to internalize precursor IL-1alpha via the FGFR trafficking pathway in HUVEC populations of pretreated with IRAP. This novel approach should not only enable them to establish the intracellular mechanism of precursor IL-1alpha as a modifier of HUVEC senescence and quiescence but may also prove valuable as a general method of importing other proteins and their mutants into recalcitrant human cells. Thus, the investigators propose to use this and more conventional gene transfer methods with ECV cells to understand the molecular basis for the repression of Src tyrosine phosphorylation in the senescent HUVEC phenotype. These studies should yield a better understanding of FGF-1 and precursor IL-1alpha signaling in HUVEC populations and firmly establish precursor IL-1alpha as a modifier of HUVEC senescence.
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