Formation of ceramide-enriched domains in lipid particles enhances the binding of apolipoprotein E.

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.

Ceramide in lipid particles enhances heparan sulfate proteoglycan and low density lipoprotein receptor-related protein-mediated uptake by macrophages.

Arterial wall sphingomyelinase (SMase) has been proposed to be involved in atherogenesis. SMase modification of lipoproteins has been shown to occur in atherosclerotic lesions and to facilitate their uptake by macrophages and foam cell formation. To investigate the mechanism of macrophage uptake enhanced by SMase, we prepared lipid emulsions containing sphingomyelin (SM) or ceramide (CER) as model particles of lipoproteins. SMase remarkably increased the uptake of SM-containing emulsions by J774 macrophages without apolipoproteins. The emulsion uptake was negatively correlated with the degree of particle aggregation by pretreatment with SMase, whereas the uptake of CER-containing emulsions was significantly larger than SM-containing emulsions, indicating that enhancement of uptake is due to the generation of CER molecules in particles but not to the aggregation by SMase. Heparan sulfate proteoglycans (HSPGs) and low density lipoprotein receptor-related protein (LRP) were crucial for CER-enhanced emulsion uptake, because heparin or lactoferrin inhibited the emulsion uptake. Confocal microscopy also showed that SMase promoted both binding and internalization of emulsions by J774 macrophages, which were almost abolished by lactoferrin. Apolipoprotein E further increased the uptake of CER-containing emulsions compared with SM-containing emulsions. These findings suggest the generation of CER in lipoproteins by SMase facilitates the macrophage uptake via HSPG and LRP pathways and plays a crucial role in foam cell formation. Thus, CER may act as an important atherogenic molecule.

Effects of plasma apolipoproteins on lipoprotein lipase-mediated lipolysis of small and large lipid emulsions.

Large (ca. 120 nm) and small (ca. 35 nm) emulsions consisting of triolein (TO) and phosphatidylcholine (PC) were prepared as the primary protein-free models of chylomicrons and their remnants, respectively. Lipoprotein lipase (LPL)-mediated lipolysis of emulsion TO was retarded in chylomicron-free human plasma compared with the hydrolysis activated by isolated apolipoprotein C-II (apoC-II). In 30% plasma, free fatty acid (FFA) release rate was higher for large emulsions than for small ones, while both emulsions were hydrolyzed at similar rates in the presence of isolated apoC-II. Isolated apolipoprotein C-III (apoC-III) or apolipoprotein E (apoE) worked as LPL-inhibitor of the lipolysis activated by apoC-II. It was also observed that apolipoprotein A-I (apoA-I) showed distinct inhibitory effects on the lipolysis of large and small emulsions: more effective inhibition for small emulsions. Kinetic analyses showed that K(m)(app) and V(max)(app) for the lipolysis of emulsions were lower in the presence of 30% plasma than isolated apoC-II. ApoA-I also markedly decreased K(m)(app) and V(max)(app) for LPL-catalyzed hydrolysis of both emulsions. In chylomicron-free serum, the density of bound apoA-I at small emulsion surfaces was about three fold greater than large emulsion surfaces, but the binding densities of apoC-II, apoC-III and apoE were less for small emulsion surfaces than for large ones, suggesting that apoA-I preferentially binds to small particles and displaces other exchangeable apolipoproteins from particle surfaces. These results indicate that, in addition to the well known inhibitory effects of apoC-III and apoE, apoA-I in plasma regulates the lipolysis of triglyceride (TG)-rich emulsions and lipoproteins in a size-dependent manner.

Effects of sphingomyelin on apolipoprotein E- and lipoprotein lipase-mediated cell uptake of lipid particles.

It has been reported that human plasma sphingomyelin (SM) levels are positively and independently related to coronary artery disease. The lipoprotein surface is mainly formed by phosphatidylcholine (PC) and SM together with cholesterol and apolipoproteins. However, the influence of SM on the cell uptake of triglyceride-rich lipoproteins and remnants is poorly understood. To clarify the role of SM in lipoprotein uptake, we prepared lipid emulsions containing triolein, PC and SM as model particles of lipoproteins. Apolipoprotein E (ApoE) binding studies revealed that incorporation of SM into the emulsion surface reduced the binding capacity of apoE without changing the affinity. Surface SM reduced apoE-mediated uptake of emulsions by HepG2 cells because of the decreased amount of binding apoE. Apolipoproteins C-II and C-III inhibited the apoE-mediated uptake of SM containing emulsions more effectively. The stimulatory effect of lipoprotein lipase (LPL) on emulsion uptake was decreased by replacing surface PC with SM. These results suggest that SM-induced changes in the binding properties of apolipoproteins and LPL correlate with decreased hepatic uptake of lipid particles.