Cholesterol crystal nucleation from enzymatically modified low-density lipoproteins: Combined effect of sphingomyelinase and cholesterol esterase
ABSTRACT An assay detecting and quantifying cholesterol nucleation from low-density lipoproteins has been established. Förster resonance energy transfer between dehydroergosterol and dansylated lecithin becomes significantly alleviated as a consequence of conucleation of dehydroergosterol and cholesterol. The assay, in combination with dynamic light scattering, absorbance spectroscopy, and fluorescence microscopy, can be used to study aggregation and nucleation in model blood systems. Human plasma LDL was labeled with dehydroergosterol and dansylated lecithin by incubation with donor multilamellar liposomes and isolated by centrifugation. Exposure of labeled LDL (0.5 mg/mL of total lipids) to sphingomyelinase (0.0-0.2 unit/mL) led to modest particle aggregation but produced no changes in energy transfer and no crystallization. However, addition of sphingomyelinase produced significant particle aggregation, nucleation, and crystallization, in a dose-dependent fashion, in samples that were previously treated with the enzyme, cholesterol esterase (0.2 unit/mL). The combination of cholesterol esterase and sphingomyelinase led to a significant alleviation of energy transfer, which preceded by 24 h the appearance of fluorescent, microscopic sterol crystals. These results point to a synergistic effect between cholesterol esterase and sphingomyelinase, suggesting that mere aggregation of LDL is insufficient to promote nucleation, and crystal formation likely proceeds in the intracellular space after LDL uptake by macrophages.
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ABSTRACT: We have set-up a scanning near-field infrared microscope (SNIM) with widely tunable lasers for label-free molecule identification by infrared spectral fingerprints. The lateral resolution is as low as 30 nm corresponding to the nanotip curvature radius. We obtained infrared spectra from nanoscale sections of self-assembled monolayers (SAM) with volumes as small as 0.01 attoliter corresponding to less than 30000 molecules. Spectroscopy of lipid bilayer stacks on mica revealed nanoscale near-field depth resolution of 80 to 120 nm. We discuss combined systems of membrane proteins and lipids on SAM supports approaching cell like membrane structures. We report on the progress of the set-up of an infrared and terahertz near-field microscope for the new synchrotron beamline at ANKA for full spectral nanoscale information retrieval.Proceedings of SPIE - The International Society for Optical Engineering 02/2009; 7188:71880I. DOI:10.1117/12.808276 · 0.20 Impact Factor
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ABSTRACT: Liposomes containing rigid and bulky lipid molecules such as sterols and pyrene‐labeled lipids can exhibit biphasic changes in membrane properties at several specific mole fractions predicted by the theory of lipid regular distributions (e.g., superlattices) in the plane of the membrane. This phenomenon has been observed in two‐component as well as multicomponent liquid‐crystalline liposomal membranes. The extent of sterol regular distribution plays a role in drug partitioning into membranes, the activities of surface‐acting enzymes such as cholesterol oxidase and phospholipase A2, and in free‐radical‐induced sterol oxidation. This article summarizes the original fluorescence studies of lipid superlattices, reviews the recent findings in this area, and discusses the current controversial issues related to lipid regular distributions.Soft Materials 05/2004; 2:85-105. DOI:10.1081/SMTS-200056098 · 1.74 Impact Factor
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ABSTRACT: Atherosclerotic lesions are often hypoxic and exhibit elevated lactate concentrations and local acidification of the extracellular fluids. The acidification may be a consequence of the abundant accumulation of lipid-scavenging macrophages in the lesions. Activated macrophages have a very high energy demand and they preferentially use glycolysis for ATP synthesis even under normoxic conditions, resulting in enhanced local generation and secretion of lactate and protons. In this review, we summarize our current understanding of the effects of acidic extracellular pH on three key players in atherogenesis: macrophages, apoB-containing lipoproteins, and HDL particles. Acidic extracellular pH enhances receptor-mediated phagocytosis and antigen presentation by macrophages and, importantly, triggers the secretion of proinflammatory cytokines from macrophages through activation of the inflammasome pathway. Acidity enhances the proteolytic, lipolytic, and oxidative modifications of LDL and other apoB-containing lipoproteins, and strongly increases their affinity for proteoglycans, and may thus have major effects on their retention and the ensuing cellular responses in the arterial intima. Finally, the decrease in the expression of ABCA1 at acidic pH may compromise cholesterol clearance from atherosclerotic lesions. Taken together, acidic extracellular pH amplifies the proatherogenic and proinflammatory processes involved in atherogenesis. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.