Wednesday, February 5, 2025
2/5/2025
Project Summary The excessive accumulation of cholesterol in vascular macrophages is regarded as a leading factor in the development of vascular inflammation, plaque instability and clinical manifestations of atherosclerosis. However, recent advances in single cell analyses and our studies suggest that inflammatory genes are predominantly expressed in macrophages that accumulate cholesterol in the plasma membrane rather than in lipid droplet- laden macrophage foam cells. Cholesterol and many receptors governing inflammatory responses colocalize in the ordered plasma membrane microdomains, often designated as lipid rafts. Upon activation, lipid raft resident and recruited proteins assemble and initiate signaling cascades leading to inflammation. We introduced the term inflammarafts, defined as enlarged, clustered lipid rafts harboring activated receptors and adaptor molecules and serving as a scaffold to organize cellular inflammatory responses. We found inflammarafts to be surprisingly stable in macrophages isolated from atherosclerotic lesions. We further identified apoA-I binding protein (AIBP) as a key regulator of cellular cholesterol metabolism, which can selectively target inflammarafts via its binding to TLR4, without disrupting physiological lipid rafts. In preliminary studies, we found that non-foamy macrophages but not macrophage foam cells expressed inflammarafts, which correlated with atherosclerosis burden. In addition, hypercholesterolemic AIBP deficient mice, which we created, developed exacerbated atherosclerosis. In contrast, the AAV-mediated expression of a secreted form of AIBP in the liver reduced atherosclerosis. In addition, mitochondria in AIBP-deficient cells were morphologically distorted, with a characteristic hyper- branched and cupped shape, typically associated with oxidative stress. The goal of this proposal is to delineate mechanisms governing differential inflammaraft dynamics and related mitochondrial dysfunction in macrophage foam cells and in non-foamy macrophages in atherosclerosis. Specifically, we will test the hypothesis that reversal of inflammarafts in non-foamy macrophages reduces vascular inflammation and is atheroprotective. The hypothesis will be tested using genetic and AAV tools to achieve constitutive, macrophage-specific and/or inducible loss-of-function or gain-of function of cholesterol transporters ABCA1/G1 and/or AIBP and its variant that does not bind TLR4. In addition, we will test the hypothesis that AIBP protects macrophages from mitochondrial dysfunction and oxidative stress in atherosclerosis. We will examine mitochondrial architecture and function in macrophages with loss- and gain-of-function of different forms of AIBP and/or ABCA1/G1. Methods will include serial block-face scanning electron microscopy (EM) and multi-tilt EM tomography, along with measures of bioenergetics by Seahorse. To assess the relevance of our hypotheses and the findings to human cardiovascular disease, we will characterize macrophage inflammarafts and mitochondrial dysfunction in coronary arteries from explanted hearts of patients with heart failure due to atherosclerotic coronary artery disease or due to non-ischemic cardiomyopathy, which undergo heart transplant surgery.
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