Moderate ethanol consumption protects against coronary artery disease by as yet unclear mechanisms. Endothelial dysfunction with over expression of adhesion molecules and increased leukocyte-endothelial adherence occurs early in atherosclerosis. Our preliminary studies in cultured endothelium show that chronic exposure (72 h) to physiologically relevant concentrations of ethanol (25 and 50 mM) causes a dose-dependent increase in the nuclear translocation of NF-kB. This is associated with reduced NF-kB response to subsequent stimulation by tumor necrosis factor alpha (TNF) and with decreased expression of vascular cell adhesion molecule 1 (VCAM-1), an NF-kB-regulated adhesion molecule that is over expressed early in atherosclerosis. The central hypothesis of this proposal is that clinically relevant concentrations of ethanol disrupt the NF-kB pathway by inducing the nuclear translocation of inhibitory NF-kB proteins or by interfering with the function of activator NF-kB proteins. This results in inhibition of subsequent NF-kB responses to activator cytokines which may make the endothelium less susceptible to activation and less capable of triggering the chain of events that initiates atherosclerosis. Therefore, an extension of our hypothesis is that the decreased expression of NF-kB-dependent genes induced by ethanol reduces adhesion molecule and chemokine expression which in turn results in decreased monocyte adherence to endothelium and inhibition of atherosclerotic plaque formation. We propose four potential mechanisms for the effects of ethanol on NF-kB: (1) increased proteolysis of I-kB due to enhanced phosphorylation or proteasomal degradation; (2) decreased synthesis of I-kB; (3) decreased Bcl-3 activity resulting in increased levels of nuclear p50 homodimers; and (4) increased synthesis of NF-kB/Rel proteins. We propose to study these mechanisms by measuring NF-kB activity, the levels of NF-kB/Rel and I-kB proteins, and proteasomal activity using gel shift assays, Northern and Western blots, and in vitro processing of NF-kB precursors, respectively. We will also study the effects of ethanol on the expression of the NF-kB-regulated genes VCAM-I and macrophage colony stimulating factor by measuring their protein and mRNA levels, and performing DNA footprinting and functional promoter assays. Lastly, we will assess the effects of ethanol on monocyte-endothelial cell adhesion in vitro. This project addresses a novel and important area in alcohol research and should enhance our understanding of the pathogenesis of ethanol-induced endothelial dysfunction and its cardiovascular consequences in humans.
