Endothelial cells form a ubiquitous interface between the blood and underlying tissues. Because of their strategic position, endothelial cells contribute to many hemostatic processes. Changes in endothelial cell gene expression, for example in response to oxidized lipids or inflammatory cytokines, contribute to the pathology of atherosclerosis and septic shock. In atherosclerosis, endothelial cell activation results in the altered expression of adhesion molecules which results in recruitment of leukocytes to areas of inflammation. In septic shock, the normally anticoagulant surface of the endothelial cell may be changed to a procoagulant state by induction of tissue factor (TF), the cellular initiator of coagulation. These changes in gene expression are, in large part, coordinated by the NFkB/Rel family of transcription factors. The overall goal of this proposal is to elucidate the molecular mechanisms that regulate expression of inflammatory genes in endothelial cells. In particular, the specific aims will determine the mechanism by which elevated intracellular cAMP inhibits NFkB/Rel mediated gene expression. Firstly, inhibition of the endogenous TF, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin genes will be studied in endothelial cells exposed to TNFa. Secondly, the effect of elevated cAMP on the nuclear translocation, composition and cellular distribution of NFkB/Rel complexes will be studied. Thirdly, the hypothesis that elevated intracellular cAMP inhibits the functional activity of NFkB/Rel proteins will be investigated. Recently, both a consensus kB-site and the TF kB-like site were shown to be necessary for cAMP inhibition. In addition, transactivation by p65 was inhibited by elevated intracellular cAMP. Fourthly, to test the hypothesis that changes in protein phosphorylation alter the function of NFkB/Rel proteins, the pattern of phosphorylation in response to TNFa, in the presence or absence of agents that elevate intracellular cAMP, will be determined. Finally, factors which modulate the activity of NFkB/Rel proteins will be identified. Elucidation of the mechanism by which elevated intracellular cAMP inhibits gene expression in endothelial cells should allow the development of effective strategies for prevention and treatment of thrombotic and vascular diseases. The goal of this proposal is to elucidate molecular mechanisms that regulate expression of inflammatory genes in endothelial cells, with particular emphasis on the mechanism whereby elevated intracellular cAMP inhibits NF-KB/Rel mediated gene expression. The effects of cAMP on three genes, TF, VCAM, and E-selectin will be evaluated, with reference to the relationship to nuclear translocation, composition and cellular distribution of NF-KB/Rel complexes.The effects of intracellular cAMP on the functional activity and phosphorylation of NFKB/Rel proteins will be investigated. A yeast two hybrid system will be used to identify additional factors which interact with NFKB.
