Radiation damage to the microvasculature is a major component int he pathogenesis of late radiation injury to normal tissues, and endothelial cells represent the most sensitive targets for radiation in the vessel wall. Despite the central role that endothelial cells play in the expression of radiation damage in vivo, only relatively little is known about their radiobiology. The goal of this research is to investigate the mechanisms by which fibroblast growth factor (FGF) induces the repair of potentially lethal radiation damage (PLDR) in cultured bovine aortic endothelial cells (BAEC). We propose the hypothesis that FGF, which under normal conditions regulates the growth and differentiation of endothelial cells, initiates in irradiated endothelial cells receptor mediated signals that lead to repair of radiation damage. This hypothesis is based on results from more recent experiments which showed that FGF, given either in free form or as bound to basement membrane-like extracellular matrix (ECM), increases the survival of irradiated plateau (mostly G0) phase BAEC by stimulating a repair process, classified as the alpha type of PLDR. The experiments are designed to further characterize the induction of PLDR by FGF in endothelial cells and to study its mechanism adopting two major approaches. the first involves the use of known inhibitors of PLDR as probes to identify and study specific biochemical mechanisms involved in the putative FGF-dependent alpha-PLDR pathway. It will also address the question whether repair of DNA strand breaks underlies the mechanism of FGF-dependent PLDR, and whether inhibitors of alpha-PLDR modulate this process. The second approach addresses the initial events in the alpha- PLDR pathway in relation to other known effects of FGF on G0 phase endothelial cells, such as its ability to initiate receptor mediated signals that are transduced to the nucleus, which facilitate endothelial cell emergence from the quiescent state into a cell cycling mode and the activation of DNA metabolism, leading to DNA replication. the experiments are designed to study the cell cycle dependence of PLDR and DNA strand break repair during the FGF stimulated transition from G0 to G1 phase, and to explore the FGF signaling transduction mechanisms that operate in irradiated endothelial cells as the initial events in the alpha-PLDR pathway. The preliminary data suggest that the FGF effects on mitogenesis and alpha-PLDR operate in endothelial cells via different pathways, and that the alpha-PLDR pathway may involve an interaction between the protein tyrosine kinase (PK) mechanism and the phospholipase C-protein kinase C (PLC-PKC) mechanism. This hypothesis will be tested. The successful demonstration of the initial events involved in the putative alpha-PLDR pathway, combined with the information derived from the experiments with the PLDR inhibitor probes, and the studies on the effects of FGF on DNA strand break repair, may contribute to the understanding of the biochemical mechanisms and molecular basis of PLDR. The experiments planned in this research are based on the use of a new in vitro system of BAEC, ECM and FGF that closely resembles the vascular intima in vivo, and permits the study of factors that modulate the radiation response of endothelial cells under conditions relevant to the in vivo effects of radiation on the microvasculature.
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