ABSTRACT
To investigate the effect of apolipoprotein B (apoB) on cell viability, we used lipid-free apoB as a model for denatured apoB. Lipid-free apoB had cytotoxicity to J774 macrophages, CHO cells and HepG2 cells, whereas apoB bound to low density lipoprotein (LDL) and lipid-free apolipoprotein A-I had no effect on cell viability. Lipid-free apoB induced apoptosis in J774 macrophages assessed by caspase-3 activation and annexin V binding. LDL receptor, heparan sulfate proteoglycans, and class A scavenger receptor were involved in the binding/uptake of lipid-free apoB, but lipid-free apoB binding/uptake by the cells did not correlate with cytotoxicity. Lipid-free apoB disrupted the lipid bilayer of large unilamellar vesicles containing calcein. We evaluated the interaction between apoB and cellular membrane by monitoring the change in intracellular Ca(2+) concentration using Fura-2, and found that lipid-free apoB rapidly disrupted the cellular membrane in the absence or presence of the inhibitors for cellular binding/uptake mediated by the receptors. Therefore, it is suggested that lipid-free apoB induces cell death by disturbance of the plasma membrane. In addition to other lipid component in modified LDL, apoB itself has an ability to induce apoptosis and plays a crucial role in the development of atherosclerotic lesions.
Subject(s)
Apolipoproteins B/pharmacology , Animals , Annexin A5/metabolism , Antibodies/pharmacology , Apolipoproteins B/chemistry , Calcium Signaling/drug effects , Caspase 3/metabolism , Cell Death/drug effects , Cell Line , Cell Shape/drug effects , Cell Survival/drug effects , Circular Dichroism , Enzyme Activation/drug effects , Fluoresceins/metabolism , Heparan Sulfate Proteoglycans/metabolism , Heparin/pharmacology , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , L-Lactate Dehydrogenase/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/enzymology , Mice , Protein Binding/drug effects , Protein Structure, Secondary , Scavenger Receptors, Class A/metabolismABSTRACT
Chylomicron remnants have been suggested to be involved in the development of atherosclerosis. To investigate the mechanisms of chylomicron remnant-induced atherosclerosis, we prepared cholesterol (Chol)-containing emulsion particles as models for chylomicron remnants. Chol markedly increased the apolipoprotein E (apoE) binding maximum of emulsions without changing the binding affinity and thereby promoted emulsion uptake by J774 macrophages. Fluorescence measurements showed that Chol increased acyl chain order and head group hydration of the surface phospholipid (PL) layer of emulsions. The binding maximum of apoE was closely correlated with the hydration and the increase in the PL head group separation at the emulsion surface. From experiments using inhibitors for lipoprotein receptors, heparan sulfate proteoglycans and low density lipoprotein receptor-related protein were found to be the major contributors to the uptake of Chol-containing emulsions. Trypan blue dye exclusion revealed that the uptake of Chol-containing emulsions induced cytotoxicity to J774 macrophages. This study proposes a mechanism of atherosclerosis induced by chylomicron remnants.
Subject(s)
Apolipoproteins E/metabolism , Cholesterol/pharmacology , Chylomicrons/pharmacology , Emulsions , Macrophages/drug effects , Animals , Apolipoproteins E/chemistry , Apoptosis/drug effects , Cell Line , Cell Survival/drug effects , Cholesterol/chemistry , Chylomicron Remnants , Chylomicrons/metabolism , Heparan Sulfate Proteoglycans/metabolism , Humans , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Macrophages/cytology , Mice , Surface PropertiesABSTRACT
We investigated the interaction between apolipoprotein E (apoE) and ceramide (CER)-enriched domains on the particles, by using lipid emulsions containing sphingomyelin (SM) or CER as model particles of lipoproteins. The sphingomyelinase (SMase)-induced aggregation of emulsion particles was prevented by apoE. CER increased the amount of apoE bound to emulsion particles. The confocal images of CER-containing large emulsions with two fluorescent probes showed three-dimensional microdomains enriched in CER. SMase also induced the formation of CER-enriched domains. We propose apoE prefers to bind on CER-enriched domains exposed on particle surface, and thus inhibits the aggregation or fusion of the particles.
