This is a competing continuation application to continue our work to understand the relationship between apoE synthesis/secretion, and macrophage function. The application is based on three hypotheses. Hypothesis 1: There are several common and naturally occurring isoforms of apoE, which occur in the American population, and which have been shown to have differential effects on atherosclerosis in vivo. We have already shown that expression of the most common occurring isoform, apoE3, in macrophages modulates sterol efflux, binds to cell surface proteoglycans, generates a heterogeneous cell surface pool that is important for sterol efflux, recycles at the cell surface, and is regulated post-translationally by sterols. We hypothesize that the differential effects of naturally occurring isoforms of apoE on atherogenesis are related to different properties with respect to these functions in the macrophage. The function of selected, artificially generated mutants, with potential to be informative from the structure-function point-of-view, will also be evaluated. Hypothesis 2: We propose that different cellular pools are utilized for efflux by the endogenous expression of apoE, compared to the exogenous addition of apoE. As a reflection of this, we propose that the composition and function of the particles generated by these alternative ways of introducing apoE will be different. Hypothesis 3: We propose that endogenously synthesized apoE, and ABCA1 mediate sterol efflux via separate pathways; but that regulation of ABCA1 and apoE expression are integrated at post-translational loci. The experiments to address these three hypotheses are contained in three Specific Aims; each of which builds on a body of preliminary data that is summarized in the application or included in the appendix. The information obtained from these investigations should further elucidate the role of endogenously expressed apoE in maintaining normal macrophage functions; particularly those functions that relate to normal vessel wall homeostasis and atherogenesis. This information will significantly impact our understanding and approach to an important human disease because of the important role that has been demonstrated for macrophage-derived apoE in preventing atheroscierosis, and maintaining normal vessel wall homeostasis.
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