Functional Hierarchy of Remnant Lipoprotein Receptors

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DESCRIPTION (provided by applicant): The atheroma macrophage lives in a peculiar environment, where cholesterol excesses trigger diverging signals of active phagocytosis and inflammatory bursts. During the previous cycles of this grant, we have focused on two proteins that have both cooperative and independent functions on cellular cholesterol trafficking and regulation of inflammation, apoE and LRP1. ApoE is an LXR-regulated protein, highly expressed in macrophages under conditions of cholesterol loading, with the ability to drive cholesterol efflux from the cell and with direct anti-atherogenic effects. However, apoE can also associate with atherogenic lipoproteins and then bind to internalizing receptors such as LDLR and LRP1, thus driving cholesterol back into the cell. LRP1, a receptor for multiple ligands, acts in the liver as a back-up system for the LDLR to clear remnant lipoproteins. Hepatic LRP1 needs locally produced apoE to clear incoming remnants already enriched in plasma-derived apoE, whereas LDLR clears apoE-containing lipoproteins even in the absence of locally produced apoE. With the working hypothesis that apoE and LRP1 interact more intricately than in a relation between lipoprotein ligand and its internalizing receptor, we moved to an experimental stage where LDLR is not the dominant lipoprotein receptor and where the interaction may have complex biological consequences, the atheroma macrophage. We demonstrated that absence of LRP1 in macrophages increases atherogenesis, a paradoxical effect given that it also reduced internalization of remnant lipoproteins and increased expression of apoE. These data suggest both that the effect of LRP1 is not linked to bulk cholesterol transport and that the vascular effect of apoE is mediated by an interaction with LRP1. We were surprised to determine that the latter is not true, as the deletion of apoE aggravated the atherogenic phenotype of LRP1-/- macrophages, thus suggesting that these proteins have independent and additive effects that regulate vascular homeostasis. The plaques of apoE-/-/LRP1-/- mice showed a uniquely severe pattern of macrophage apoptosis with a deficit in the internalization of apoptotic bodies by viable phagocytes. An effect unique to LRP1 was noted on the induction of inflammatory responses, as mice carrying LRP1-/- macrophages showed significantly higher numbers of circulating and splenic pro-inflammatory Ly6chi monocytes and arterial wall Ly6chi and CCR2 positive macrophages. Another effect unique to LRP1 was noted on the regulation of prosaposin trafficking. Prosaposin leads to the formation of the saposins (sphingolipid activator proteins), critical for processing and clearance of lysosomal and membrane sphingolipids. This pathway is intertwined with the autophagic machinery, recently shown to regulate cholesterol efflux from foam cells, and with the inflammatory response. The proposed studies aim at characterizing the molecular pathways responsible for the exaggerated cell death, inflammation, and dysfunctional clearance of cell debris caused by the absence of LRP1. We strive to understand the factors that regulate bulk plaque regression.