Epidemiologic studies indicate that moderate alcohol consumption (1-2 drinks/day) reduces the risk of cardiovascular mortality and that this cardioprotective benefit may be mediated, in part, by an increase in fibrinolysis. Endothelial cells (ECs) synthesize t-PA, u-PA, PAI-1 and Receptors (Rs) for PA and plasminogen (Pmg) (PARs PmgRs) and maintain normal hemostasis and fibrinolysis by activating EC-bound Pmg through the regulated synthesis and complex interactions of these components. Alterations in these EC components /interactions by systemic factors, i.e. alcohol will promote either thrombosis or facilitate clot lysis. The investigators have shown that low ethanol (<0.1%) induces a biphasic short (<30 min) and long term sustained (12 hr) increase in cultured EC fibrinolysis. The overall goal of these revised studies is ti identify/define the molecular regulatory mechanism underlying low ethanol-induced effects on the activity, interaction, and expression of cultured human EC-produced components (Pas and Rs) resulting in rapid short vs sustained long term increases in EC-fibrinolytic activity. Studies will include kinetic analysis of short-(no Pas Rs synthesis ) vs long term (new Pas/Rs synthesis) EC-bound Pmg activation (Aim 1); binding activity (Kd, Bmax) and changes in u-PAR, t-PAR, and PmgRs mRNAs (using antisens probes for candidate Rs for t-PA [annexinII] and Pmg [annexin II and alpha -enolase] and RPAs), including determination of potential transcriptional regulation of these R types by ethanol (transcription run-on assay) (Aim 2) ; and finally identification of ethanol responsive regions in the promoter and 5-flanking regions of the t-PA and u-PA genes, including initial identication of their respective ethanol-inducible transcription factors (promoter deletion analysis, transient transfection, EMSA) (Aim 3). Results gleaned from these studies will provide significant new insights into our understanding of the mechanisms underlying the upregulation of EC-mediated fibrinolytic activity by low ethanol, at both, the molecular and gene levels. Increased EC-fibrinolysis will substantially decrease the risk for thrombosis, CAD and associated atherothrombotic events leading to MI and will provide a well defined molecular basis to explain, in part, the cardioprotective benefit and reduced risk of cardiovascular mortality associated withe moderate alcohol consumption.
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