: Ischemic and inflammatory diseases target the vascular endothelium and are marked by increased levels of cytokines, intense oxidative stress, and increased vascular permeability. The precise nature of the relationship between these three features of vascular diseases is ill defined, however, impeding efforts to treat these common syndromes. Oxidants have only recently been appreciated as participating in physiologic signaling processes, particularly those initiated by TNF-alpha, although the source and targets of such oxidants are not known. In simple in vitro systems, the evanescent nature of oxidants renders their effects highly site-specific, such that their site of origin generally dictates their molecular target. Recent studies have also revealed that signal specificity, particularly in the MAP Kinase system, also depends on proper colocalization of signaling elements. Our preliminary data implicate a phagocyte oxidase-like complex in the TNF-alpha induced activation of c-Jun amino terminal kinase (JNK), a MAP kinase whose active form is associated with the cytoskeleton, in human endothelial cells. We have cloned p47phox, an important signal-receiving adapter protein for the oxidase, from a human endothelial cell library, and found that the p47phox protein is also localized to the cytoskeleton. Further, we have made and screened a HUVEC GAL4 library for binding partners of p47phox, and have recovered several important genes detailed in the body of the grant. We hypothesize that inflammatory signaling by cytokines and growth factors in vascular cells involves site-specific production of oxidants by a phagocyte oxidase-like complex, which leads to downstream activation of JNK and NF-KB. We further propose that the oxidase is tethered to cytoskeleton-associated elements through specific interactions with signaling proteins. Our overall objectives will be to investigate the nature of p47phox interactions with three potential binding targets, and to explore the functional significance of these interactions by examining upstream and downstream signaling events. Through a more detailed understanding of cytokine-induced proinflammatory events, more opportunities will arise for designing specific interventions against ischemic and inflammatory vascular diseases.
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