Extracellular Lipids Accumulate in Human Carotid Arteries as Distinct Three-Dimensional Structures and Have Proinflammatory Properties.

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified in vitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.

The angiopoietin receptor Tie2 is atheroprotective in arterial endothelium.

Leukocytes and resident cells in the arterial wall contribute to atherosclerosis, especially at sites of disturbed blood flow. Expression of endothelial Tie1 receptor tyrosine kinase is enhanced at these sites, and attenuation of its expression reduces atherosclerotic burden and decreases inflammation. However, Tie2 tyrosine kinase function in atherosclerosis is unknown. Here we provide genetic evidence from humans and from an atherosclerotic mouse model to show that TIE2 is associated with protection from coronary artery disease. We show that deletion of Tie2, or both Tie2 and Tie1, in the arterial endothelium promotes atherosclerosis by increasing Foxo1 nuclear localization, endothelial adhesion molecule expression and accumulation of immune cells. We also show that Tie2 is expressed in a subset of aortic fibroblasts, and its silencing in these cells increases expression of inflammation-related genes. Our findings indicate that unlike Tie1, the Tie2 receptor functions as the dominant endothelial angiopoietin receptor that protects from atherosclerosis.

Cholesterol loading suppresses the atheroinflammatory gene polarization of human macrophages induced by colony stimulating factors.

In atherosclerotic lesions, blood-derived monocytes differentiate into distinct macrophage subpopulations, and further into cholesterol-filled foam cells under a complex milieu of cytokines, which also contains macrophage-colony stimulating factor (M-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF). Here we generated human macrophages in the presence of either M-CSF or GM-CSF to obtain M-MØ and GM-MØ, respectively. The macrophages were converted into cholesterol-loaded foam cells by incubating them with acetyl-LDL, and their atheroinflammatory gene expression profiles were then assessed. Compared with GM-MØ, the M-MØ expressed higher levels of CD36, SRA1, and ACAT1, and also exhibited a greater ability to take up acetyl-LDL, esterify cholesterol, and become converted to foam cells. M-MØ foam cells expressed higher levels of ABCA1 and ABCG1, and, correspondingly, exhibited higher rates of cholesterol efflux to apoA-I and HDL2. Cholesterol loading of M-MØ strongly suppressed the high baseline expression of CCL2, whereas in GM-MØ the low baseline expression CCL2 remained unchanged during cholesterol loading. The expression of TNFA, IL1B, and CXCL8 were reduced in LPS-activated macrophage foam cells of either subtype. In summary, cholesterol loading converged the CSF-dependent expression of key genes related to intracellular cholesterol balance and inflammation. These findings suggest that transformation of CSF-polarized macrophages into foam cells may reduce their atheroinflammatory potential in atherogenesis.