This renewal application requests support to study the mechanisms involved in the regulation of human endothelial cell senescence in vitro. Prior observations from our laboratory and others have established that human endothelial cell growth is regulated by the signal-less heparin-binding fibroblast growth factor (HBGF) prototypes while human endothelial cell quiescence is regulated by polypeptide cytokines including interleukin (IL)-1alpha. Recent efforts have suggested that the repression of translation of IL-1alpha significantly extends the lifespan of the human umbilical vein endothelial cell (HUVEC) in vitro. These studies also suggest that the mechanism utilized by IL-1alpha to regulate HUVEC senescence involves the potential function of the signal sequence-less IL- 1alpha precursor to act as an intracellular modifier of the non-terminal HUVEC differentiation pathway. Thus, two specific aims are proposed to further define the role of IL-1alpha in HUVEC senescence. The first specific aim proposes to study the relevance of these observations to other types of human endothelial cells in vitro and are a natural extension of our prior research effort. These studies are straightforward and include the use of cell and molecular methods to define correlates between human endothelial cell senescence in vitro and the in vivo age of the donor. Because IL-1alpha is a potent inflammatory signal in vivo, these studies may provide insight into basic mechanisms involved in the regulation of vascular disease in the elderly. The second specific aim is more speculative because it is limited by our knowledge of the mechanism used by signal-less cytokines and growth factors to regulate cellular function. Our long-term goal is to understand how the signal-less IL-1alpha precursor regulates HUVEC function. Using molecular methods, we propose to identify a putative nuclear translocation signal within the structure of the precursor. In addition, we will attempt to identify and characterize the structure of intracellular polypeptides that are able to interact with the IL-1alpha precursor. While we suggest that IL-1alpha may act as an intracellular modifier of HUVEC quiescence and senescence, we also recognize that an alternative secretory pathway may exist and we propose to study this issue using our recent observation that heat-induced binding proteins may act as a vehicle for the secretion of HBGF-1. We anticipate that these efforts may provide insight into the mechanism(s) used by intracellular IL-1alpha to regulate human endothelial cell senescence in vitro and aid our understanding of the contribution of the vasculature to the aging process in vivo especially when the pathophysiology is exaggerated by inflammation and angiogenesis.
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