. Atherosclerotic cardiovascular disease remains the leading cause of death in our society. The development of atherosclerotic lesions fits the model of chronic inflammation in response to injury. Because the interior of the atherosclerotic lesion is separated from the vessel lumen, there is an extravascular inflammatory microenvironment therein. Monocytes are essential participants in both the initiation and evolution of these lesions, which are also marked by the presence of fibrin. Monocytes exposed to inflammatory stimuli such as LPS rapidly produce high levels of Plasminogen Activator Inhibitor Type 2 (PA-2), a molecule that fosters an antifibrinolytic microenvironment and allows for the persistence and maturation of fibrin deposits. Tumor necrosis factor (TNF), also produced by stimulated monocytes, may, in an autocrine manner, induce these cells to produce high levels of PAI-2 over long periods of time. PAI-2 is not inactivated by the oxidative mediators present at an inflammatory site as would be PAI-1, and PAI-2 (but not PAI-1) is produced by monocytes within the inflammatory focus, so changes in its production can be rapid and in direct response to changes in the immediate local situation. PAI-2 protein production is directly related to levels of PAI-2 mRNA, which are controlled to some extent by inducible changes in their rate of degradation. In some, although PAI-2 seems important in maintaining the anti-fibrinolytic environment of extravascular inflammatory lesions, little is known about the physiological regulation of its production. In this application we therefore propose to: (1) Determine the role of TNF in stimulating a sustained increase in monocyte production of PAI-2; (2) Determine the intracellular signalling mechanisms operative in the monocyte PAI-2 responses to LPS and TNF (i.e., does any stimulus which increases PAI-2 production trigger the same intracellular signals, or can different stimuli cause activation of different intracellular messages and yet induce the same response?); (3) Determine whether changes in mRNA stability play a significant role in regulating monocyte production of PAI-2, and if so whether the above intracellular signalling pathways have a role in determining such changes in mRNA stability.
