The mechanisms by which native Low-density Lipoprotein (LDL) perturb endothelia cell (EC) function in predisposing the vessel wall to atherosclerotic plaque formation remain unclear. In vitro systems, developed in this laboratory, examining LDL effects on EC have identified three potential mechanisms; enhanced EC generation of cytochrome P450- derived epoxyeicosatrienoic acid (EET), perturbations in EC lipid membrane dynamics, and heightened EC recruitment of monocytes and U937 cells (both referred to as MONO hereafter). These changes appear fundamentally associated with EC dysfunction and appear casually related to factors which accelerate the atherosclerotic process. Enhanced EC generation of EET is associated with an increase EC binding of MONO. LDL-treated EC metabolize EET to different products than control EC. When this altered EET metabolism is prevented with inhibitors which block P450 activity, MONO binding is restored to control levels. Thus, LDL alters two levels of EC metabolism, arachidonic acid (AA) and EET. The eicosanoids released by LDL-treated EC enhance EC affinity for MONO. It is hypothesized that this increased affinity develops at the protein level of surface cell adhesion molecule (CAM) receptor synthesis based on cycloheximide inhibition of MONO binding. The effects described for EET-and LDL-enhanced EC recruitment of MONO is not due to LDL oxidation. Thus, significant normal relationship exists between LDL-induced perturbations in oxidative metabolism of AA and recruitment of MONO. This proposal focuses on detailing how LDL alters EC metabolism of AA and EET to increase EC affinity for MONO. Experiments are designed to elucidate the mechanisms by which LDL and EET induce CAM synthesis and distribution and increase EC MONO binding. The changes are examined at the level of function (MONO) binding). CAM protein/eicosanoid measurements, and alterations at the molecular level concerning oxidative enzymes (cyclooxygenase, lipoxygenase, and P450) and CAM receptor expression. It is hypothesized that LDL induces perturbations in AA and EET metabolic pathways to generate novel inflammatory eicosanoids. In an autocrine fashion these eicosanoids enhance EC CAM expression and MONO recruitment. Modern molecular biology techniques are employed to delineate LDL's effect on EC P450 epoxygease and w/w-1 hydroxylase activities and to determine the molecular basis by which these changes develop. By understanding the details of LDL-induced alterations in EC oxidative AA and EET metabolism and determining the relationship between heightened EC EET generation and mono recruitment it may be possible to develop therapeutic interventions aimed at preventing LDL induced disturbances in EC function.
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