J. thermally induced fusion. Monoclonal antibody binding suggests possible involvement of apoB N-terminal domain in early stages of LDL fusion. LDL fusion accelerates at pH 7, which may contribute to LDL retention in acidic atherosclerotic lesions. Fusion also accelerates upon increasing LDL concentration in near-physiologic range, which likely contributes to atherogenesis. Thermal stability of LDL decreases with increasing particle size, indicating that the pro-atherogenic properties of small dense LDL do not result from their enhanced fusion. Our work provides the first kinetic approach to measuring LDL stability and suggests that lipid-lowering therapies that reduce LDL concentration but increase the particle size may have opposite effects on LDL fusion. strong class=”kwd-title” RU 58841 Keywords: LDL aggregation, fusion, and rupture; sigmoidal denaturation kinetics; LDL subclasses; acidic pH; high-order reaction; lipid-lowering therapies; atherosclerosis Low density lipoproteins (LDL, also known as bad cholesterol) are noncovalent assemblies containing hundreds of lipids and a single copy of apolipoprotein B (apoB), a 550 kDa glycoprotein that comprises over 95% of total LDL protein (1). Plasma LDL are the major vehicles for cholesterol delivery to peripheral cells (2). Plasma levels of LDL cholesterol and, particularly, apoB are the strongest predictors of atherosclerosis and its causative agents (3). In atherosclerosis, LDL lipids are deposited in the arterial intima; according to the response-to-retention paradigm (4, 5), LDL retention by the arterial matrix proteoglycans triggers a cascade of pro-atherogenic events culminating in formation of atherosclerotic plaque (6C8). These events include biochemical modifications of LDL, such as oxidation and/or hydrolysis by the resident proteases and lipases, e.g., phospholipase A2 and sphingomyelinase, which can reduce LDL affinity for the LDL receptor, increase LDL affinity for the proteoglycans, and promote LDL fusion (7C14). In addition, ionic interactions with proteoglycans reduce LDL stability and promote their fusion and rupture (i.e., release of core lipids) (15). Fusion of lipoproteins prevents their exit from the arterial intima and thereby augments their further modifications, enhances LDL uptake by the arterial macrophages, and initiates the formation of atherosclerotic lesions (9, 16). Therefore, the pro-atherogenic potential of LDL is thought to be linked to their ability to fuse (9, 10, 17). Dissecting the pathogenic pathway of LDL fusion and identifying key factors that promote or inhibit this pathway can help obtain new therapeutic targets for atherosclerosis. Structural analysis of intact and modified LDL has been limited to low resolution (16 ?) by the large size and hydrophobicity of apoB and by LDL heterogeneity (1, 18C21). Human plasma LDL consist of subclasses differing in particle diameter (20C24 nm), charge, biochemical composition, apoB conformation, and metabolic properties (17, 22C24). Among these subclasses, small dense LDL (sdLDL) are thought to be particularly pro-atherogenic due to their reduced affinity to LDL receptor, increased affinity to arterial proteoglycans, and increased susceptibility to pro-atherogenic modifications, such as oxidation (23C25). Furthermore, electronegative LDL show enhanced pro-atherogenic properties (17, 26C28) that have been linked to their increased propensity to aggregate and fuse (17). The relative fusion propensity of sdLDL versus their larger counterparts remains unknown and is addressed in this work. Although RU 58841 the detailed structural basis for LDL fusion is unclear, it is thought to result from accumulation of packing defects on LDL surface that form upon mechanical, thermal, chemical or enzymatic perturbations (9C15, 29C32). The latter include lipolysis of polar lipids, such as phosphatidylcholine or sphingomyelin, and proteolysis of apoB followed by dissociation of its proteolytic fragments (7C15, 32). We showed that thermal denaturation2 mimics in vivo LDL aggregation, fusion, and rupture (i.e., release of neutral lipids from LDL core, which coalesces into droplets) (30) (supplementary Fig. I). Thermal denaturation studies revealed that stability of LDL and other lipoproteins is determined by kinetic barriers and suggested RU 58841 that similar barriers modulate lipoprotein remodeling and fusion in vivo (30, 33C36). In fact, the products of the heat-induced LDL fusion are similar in size and morphology to LDL-derived extracellular deposits in early atherosclerotic lesions (37). To quantify LDL stability, we used a kinetic approach applied in the previous thermal stability studies of high- and very low-density lipoproteins, HDL and VLDL (34C36), and HDL subclasses (38). The application of this approach to IGFBP6 LDL has been complicated by the relatively narrow range of experimental.
