Regulation of Endothelial Lipase and HDL Metabolism by ANGPTL3

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PROJECT SUMMARY Despite currently available treatments, cardiovascular disease remains the leading cause of death in the United States. As dysregulated lipoprotein metabolism contribute to cardiovascular disease, there is a critical need to decipher the lipoprotein pathways, including those regulated by ANGPTL proteins, that drive cardiovascular pathologies. The long-term goal of the proposed research is to understand how ANGPTL proteins regulate lipid homeostasis and how these proteins become dysregulated in disease. One ANGPTL protein, the hepatokine ANGPTL3, inhibits both endothelial lipase (EL) and lipoprotein lipase, and thus regulates both plasma cholesterol and triglycerides. The objective in this application is to identify the mechanisms by which ANGPTL3 regulates EL and how this regulation affects HDL metabolism and cardiovascular disease. The central hypothesis of this study is that ANGPTL3 inhibits EL by promoting structural changes that disrupt its stability, and that this inhibition prevents the pathologic remodeling of HDL that contributes to atherosclerosis. This hypothesis will be tested by pursuing two specific aims: 1) Determine the effects of ANGPTL3-mediated EL inhibition on atherosclerosis and HDL function; and 2) Identify the mechanism by which ANGPTL3 inhibits EL. The studies in aim 1 use mice that express mutant ANGPTL3 alleles that only inhibit EL or LPL. These mice will be crossed with ApoE knockout mice to determine the respective contributions of ANGPTL3-mediated EL inhibition and LPL inhibition to atherosclerosis. These mice will also be used to determine the physiologic locations of EL inhibition using tissue phospholipase activity assays. Radioactive tracer experiments will be used to determine how inhibition of EL by ANGPTL3 alters HDL-lipid partitioning, and a variety of HDL functional assays will be used to determine how this inhibition alters the anti-atherogenic functions of HDL. In aim 2, various biochemical analyses, including phospholipase activity assays, site-directed mutagenesis, and hydrogen-deuterium exchange mass spectrometry, will be used to probe the functional interactions of ANGPTL3 with EL. The proposed studies are innovative in their use of ANGPTL3 alleles that only inhibit LPL or only inhibit EL to decouple the physiological effects of LPL and EL inhibition. The proposed studies are significant because lipoprotein homeostasis is essential for cardiovascular health. Therefore, uncovering how and where ANGPTL3 inhibits EL activity, and how this inhibition impacts HDL function, HDL metabolism, and the progression of atherosclerosis will increase our fundamental understanding of how lipoprotein metabolism is regulated. Completion of the aims outlined in this study will have a broad positive impact as lipoprotein metabolism contributes to many metabolic diseases, and thus understanding how the regulators of these pathways, including ANGPTL3, can best be therapeutically targeted may have wide-reaching implications for improved outcomes for these diseases.