During the current grant period we characterized a number of LXR target genes including Abcgl, that is thought to function as a sterol transmembrane transporter. Abcgl was shown to be expressed at high levels, in a number of cell types including macrophages, B and T cells. We used cultured cells and Abcgl-/- mice to demonstrate that ABCG1 functions to flux sterols away from the endoplasmic reticulum to exogenous sterol acceptors, such as HDL. Loss of ABCG1 was associated with massive lipid accumulation, cytokine activation, inflammation and an increase in IgM-secreting B-1 cells. Importantly, bone marrow transplantation (BMT) studies identified a role for ABCG1 in the development of atherosclerosis. In collaboration with Project 6 we also demonstrated an unexpected and novel role for ABCG1 in T cell proliferation. We now propose to use two new mouse models, Abcgl-/-apoE-/- and Abcgl-/-Rag-/- double knockout mice: i) to test the hypothesis that loss of ABCG1 attenuates the development of atherosclerosis as a result of increased apoptosis, and to then identify the apoptotic pathway/mechanism involved, ii) to test the hypothesis that loss of ABCG1 from B and T cells (and possibly B-1 cells) also affects atherosclerosis. The latter studies will utilize adoptive transfer of B and T cells into Abcgl-/-RAG-/- mice followed by BMT into Ldlr-/- recipient mice. In preliminary studies we have identified multiple oxysterols that accumulate in Abcgl- /- macrophages in vivo. We now propose to better characterize the lipids and oxysterols that accumulate and then determine the mechanism by which these specific oxysterols/lipids promote apoptosis both in vivo and in vitro and induce massive cytokine expression, especially in macrophages lacking ABCGL We will also use state-of-the-art spinning disc microscopy and fluorescent-tagged wild type or mutant ABCG1 to follow intracellular movement of the transporter as a means of understanding how ABCG1 functions to flux sterols through intracellular vesicles to the cell surface. Finally, we will use a new cell-based assay to test the hypothesis that ABCG1, but but not ABCA1, functions to promote the efflux of specific sterols away from the endoplasmic reticulum.
Lay Summary: The presence of cholesterol-loaded macrophages in the artery wall is associated with the development of atherosclerotic lesions and heart attacks. The current studies aim to explore mechanisms by which certain proteins move cholesterol out of cells and thus provide protection from inflammation and atherosclerosis.
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