Size-selective uptake of colloidal low density lipoprotein aggregates by cultured white blood cells

Abstract

This paper illustrates how principles of colloid science are useful in studying atherosclerosis. Accumulation of foam cells in the arterial intima is a key step in atherogenesis. The extent of foam cell formation is enhanced by low density lipoprotein (LDL) aggregates, and we have previously shown that the size of sphingomyelinase (Smase)-hydrolysis-induced aggregates depends directly on the concentration of ceramide generated in the LDL phospholipid monolayer, mediated by the hydrophobic effect. Here, we focus on the effect of LDL aggregate particle sizes on their subsequent uptake by macrophages. Our data show the first direct measurement of uptake as a function of aggregate size and the first direct comparison of uptake after Smase-catalyzed and vortex-mixing-mediated aggregation. Vortex-mixed aggregates with radii 20–77 nm showed maximal uptake ∼118 μg sterol/mg protein at a 53 nm intermediate size, consistent with a mathematical model describing competition between aggregate surface area and volume. Smase-treated aggregates with radii 25–211 nm also showed maximal uptake at an intermediate size, ∼58 μg sterol/mg protein for 132 nm particles, and fit a modified model that incorporated ceramide concentration expressed as aggregate size. This study shows that particle size is significant and composition may also be a factor in LDL uptake.

Antioxidants inhibit low density lipoprotein oxidation less at lysosomal pH: A possible explanation as to why the clinical trials of antioxidants might have failed

“…LDL was aggregated by incubating native LDL (2 mg protein/mL) with sphingomyelinase (Bacillus cereus, catalogue number S9396-25UN, Sigma) at 10 mU/mL ( Walters and Wrenn, 2010 ) until the attenuance (absorbance plus light scattering) at 680 nm increased from about 0.0017–0.027. Sphingomyelinase-aggregated LDL (SMase-LDL) was dialysed against a phosphate buffer containing EDTA and sterilised by membrane filtration (0.45 μm).…”

Involvement of neutral sphingomyelinase in the angiotensin II signaling pathway

“…nSMase activity was estimated as described previously by Walters and Wrenn (48). The reaction mixture contained the following components in a total volume of 200 l: 25 g renal homogenate proteins in 100 mM Tris·HCl, pH 7.4, 10 mM MgCl 2, 100 M Amplex red, 2 U/ml HRP, 0.2 U/ml choline oxidase, 8 U/ml alkaline phosphatase, and 500 M sphingomyelin.…”

Mechanistic roles of lipoprotein lipase and sphingomyelinase in low density lipoprotein aggregation

“…We have shown that LpL and Smase each cause LDL aggregation [27-28]; however they do so through different mechanisms that can both occur in lesional tissue [25-26]. Since they each modify LDL in different ways, it is likely that one mechanism could affect the other.…”

“…The Amplex Red-phosphorylcholine-coupled Smase fluorescence assay kit, purchased from Invitrogen (Carlsbad, CA), was used according to vendor instructions, and using HEPES buffer as described before [27-28]. It should be noted that the enzymatic activity observed in this work is likely less pronounced than it would be in an experimental system containing divalent cations, or in the subendothelial space or blood.…”