anti-apoB Antibody Research Articles

Prospects and limitations of antibody-mediated clearing of lipoproteins from blood plasma prior to nanoparticle tracking analysis of extracellular vesicles

ABSTRACTIntroduction: Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA).Methods: Ten platelet-free plasma (PFP) samples from healthy fasting subjects were incubated with magnetic beads coated with antibodies against apolipoprotein B-48 and B-100 (ApoB). Plasma samples were analysed with NTA before and after application of the bead procedure. Four fasting PFP samples were analysed with an ELISA specific for human ApoB to estimate the degree of removal of lipoproteins and EV array analysis was used for identification of possible EV loss.Results: The magnetic bead separation procedure resulted in a median reduction of the particle concentration in plasma by 62% (interquartile range 32–72%). The mean size of the remaining particles generally increased. ApoB concentration was reduced to a level close to the background signal, whereas a median reduction of the EV content by 21% (range 8–43%) was observed.Conclusion: Anti-ApoB antibody coated magnetic beads may hold potential for removal of lipoproteins from human PFP prior to EV measurement by NTA but some artefactual effect and EV loss may have to be endured.

Plasma lipoprotein(a) size polymorphism and function: apo(a) subunit size determines galectin-1 recognition and apoB subunit epitope masking

Polymorphism and pathological potential of human plasma lipoprotein(a) [Lp(a)] are determined solely by variation in size of its apo(a) subunit which is linked by a disulfide to an apoB subunit of invariant size. Affinity precipitation of Lp(a) from plasma employing the O-glycan-specific lectin jacalin before electrophoretic removal of adhering LDL was shown to be comprehensive without loss of lipoprotein structure. Isolation of even low abundance Lp(a) molecules by this protocol facilitated study of variations in subunit interactions as a function of Lp(a) size. Affinity of the autologous lectin galectin-1 toward Lp(a) isoforms increased with their size. Since apoB is not a ligand for galectin-1 result suggested apo(a) size as a modulator of Lp(a) adhesion in vivo to vascular cells and macrophages that express this lectin. Result may also explain the inverse relation of Lp(a) size to its rate of release from the galectin-1-rich intracellular compartments. N-Glycans and antigenic epitopes of apoB in Lp(a) were masked from recognition by the lectin concanavalin A and anti-apoB antibody respectively in proportion to the size of apo(a) . Recovery of apoB from masking upon removal of apo(a) alone from Lp(a) by dithiothreitol-mediated reduction was proportional to the size of the leaving apo(a). Since LDL receptors recognize lipoproteins through apoB epitopes results explain the reported lower binding of larger Lp(a) molecules to these receptors.

Immunopathology of desialylation: human plasma lipoprotein(a) and circulating anti-carbohydrate antibodies form immune complexes that recognize host cells.

Human plasma lipoprotein(a) [Lp(a)], the dominant lipoprotein in atherosclerotic plaques, contains an apo(a) subunit of variable size linked to the apoB subunit of a low-density lipoprotein (LDL) molecule. Circulating lipoprotein immune complexes (ICs) assayed by ELISA using microplate-coated anti-apo(a) or anti-apoB antibody for capture and peroxidase-labelled anti-human immunoglobulins as probe consisted mostly of Lp(a) despite several-fold excess of LDL over Lp(a) in plasma. Microplate coating of plasma lipoprotein IC and probing with antibodies to apo(a) and apoB also revealed negligible presence of LDL compared to Lp(a). Peanut agglutinin specific to desialylated O-glycans bound significantly more to Lp(a) recovered after urea dissociation of IC than to free Lp(a). Plasma lipoproteins separated by ultracentrifugation and desialylated by neuraminidase formed IC with naturally occurring antibodies in normal plasma. These de novo ICs agglutinated desialylated but not normal human RBC in proportion to the polyagglutinin antibody titre of plasma used, suggesting availability of multiple unoccupied binding sites on the participating antibodies even after IC formation. Agglutination was inhibitable by galactosides and decreased 4-8 fold if precursor lipoprotein was selectively depleted of Lp(a), showing agglutinating ICs were contributed mainly by desialylated Lp(a) and galactose-specific antibodies. IC was 2 fold more agglutinating if lipoproteins used contained smaller rather than larger Lp(a) molecules of the same number. Small size/high plasma concentration Lp(a) phenotype and neuraminidase-releasing diseases including diabetes are risk factors for vascular disorders. Results suggest a possible route of Lp(a) attachment to vascular cells that offer terminal galactose on surface glycans following desialylation.

Very-Low-Density Lipoprotein (VLDL)-Producing and Hepatitis C Virus-Replicating HepG2 Cells Secrete No More Lipoviroparticles than VLDL-Deficient Huh7.5 Cells

In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-β-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs.

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.

Differences between group X and group V secretory phospholipase A2 in lipolytic modification of lipoproteins

Secretory phospholipases A2 (sPLA2s) are a diverse family of low molecular mass enzymes (13–18 kDa) that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA2 (sPLA2-X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA2 (sPLA2-V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA2-X in several respects. Although sPLA2-V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA2-X. In addition, the requirement of Ca2+ for the lipolysis of LDL was about 10-fold higher for sPLA2-V than sPLA2-X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA2-V in the presence of sodium citrate, which contrasted with the potent response to sPLA2-X. Moreover, sPLA2-X, but not sPLA2-V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA2-X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (Kd = 3.1 nM) in the presence of Ca2+. Selective interaction of sPLA2-X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.

An Alternative Protein Standard to Measure Activity of LOX-1 Ligand Containing apoB (LAB) - Utilization of Anti-LOX-1 Single- Chain Antibody Fused to apoB Fragment

We have recently demonstrated that the circulating level of LOX-1 ligand containing apoB (LAB) predicts the risk of cardiovascular events; however, as is the case in other assays measuring oxidized LDL (oxLDL), chemical unstability and inter-lot variance of standard oxLDL may limit the utility of measuring LAB. This study aimed to develop an alternative protein standard that is simultaneously recognized by LOX-1 and anti-apoB antibody instead of copper-oxidized LDL. cDNAs encoding the variable regions of anti-LOX-1 monoclonal antibody were cloned from hybridomas and reorganized to express anti-LOX-1 single-chain variable fragment (Fv). cDNAs of four regions of human apoB (B1 to B4), which were reported to be epitopes of many anti-apoB antibodies, were also cloned. After confirming the respective reactivity of Fv and apoB fragments to LOX-1 and anti-apoB antibodies, cDNAs of Fv and apoB fragments were connected to express Fv-ApoB chimeric proteins. These fusion proteins were found to be recognized by both LOX-1 and anti-apoB antibodies. Among them, the fusion proteins of Fv-B1 and Fv-B3 gave saturable binding curves against immobilized LOX-1 when detected by anti-apoB antibodies. The binding curves of different Fv-B1 preparations to LOX-1 were almost identical while those of oxLDL varied among the preparations, suggesting better quality control of Fv-B1 preparations. The fusion proteins composed of Fv-form anti-LOX-1 antibody and apoB fragment are useful alternatives to copper-oxidized LDL in determining LAB, which would facilitate the application of modified LDL analyses to the clinical diagnosis and risk evaluation of cardiovascular disease.

Regulatory effects of arachidonate 5-lipoxygenase on hepatic microsomal TG transfer protein activity and VLDL-triglyceride and apoB secretion in obese mice

As 5-lipoxygenase (5-LO) is an emerging target in obesity and insulin resistance, we have investigated whether this arachidonate pathway is also implicated in the progression of obesity-related fatty liver disease. Our results show that 5-LO activity and 5-LO-derived product levels are significantly elevated in the liver of obese ob/ob mice with respect to wild-type controls. Treatment of ob/ob mice with a selective 5-LO inhibitor exerted a remarkable protection from hepatic steatosis as revealed by decreased oil red-O staining and reduced hepatic triglyceride (TG) concentrations. In addition, 5-LO inhibition in ob/ob mice downregulated genes involved in hepatic fatty acid uptake (i.e., L-FABP and FAT/CD36) and normalized peroxisome proliferator-activated receptor alpha (PPARalpha) and acyl-CoA oxidase expression, whereas the expression of lipogenic genes [i.e., fatty acid synthase (FASN) and SREBP-1c] remained unaltered. Furthermore, 5-LO inhibition restored hepatic microsomal TG transfer protein (MTP) activity in parallel with a stimulation of hepatic VLDL-TG and apoB secretion in ob/ob mice. Consistent with these findings, 5-LO products directly inhibited MTP activity and triggered cytosolic TG accumulation in CC-1 cells, a murine hepatocyte cell line. Taken together, these findings identify a novel steatogenic role for 5-LO in the liver through mechanisms involving the regulation of hepatic MTP activity and VLDL-TG and apoB secretion.

Apolipoprotein B and triacylglycerol secretion in human triacylglycerol hydrolase transgenic mice

Apolipoprotein B (apoB)-containing lipoproteins play a critical role in whole body lipid homeostasis and the pathogenesis of atherosclerosis. The assembly of hepatic apoB-containing lipoproteins, VLDL, is governed by the availability of lipids, including triacylglycerol (TG). The majority of TG associated with VLDL is derived from the hepatic cytoplasmic lipid stores by a process involving lipolysis followed by reesterification. Microsomal triacylglycerol hydrolase (TGH) has been demonstrated to play a role in the lipolysis/reesterification process. To evaluate the potential regulatory role of TGH in hepatic VLDL assembly, we developed inducible transgenic mice expressing a human TGH minigene under the control of the mouse metallothionein promoter. Induction of human TGH by zinc resulted in liver-specific expression of the enzyme associated with 3- to 4-fold increases in lipolytic activity that could be attenuated with a TGH-specific inhibitor. Augmented TGH activity led to increased secretion of newly synthesized apoB and plasma TG levels. These results suggest that increased hepatic expression of TGH leads to a more proatherogenic plasma lipid and apoB profile.

Apolipoprotein A-V association with intracellular lipid droplets

Apolipoprotein A-V (apoA-V) plays a key role in the regulation of triglyceride (TG) metabolism. Given the very low concentration of apoA-V in plasma, we hypothesized that apoA-V may influence plasma TG levels by affecting the assembly and/or secretion of apoB-containing lipoproteins. When apoA-V was overexpressed in cultured Hep3B cells, neither the amount of apoB secreted nor the density distribution of apoB-containing lipoproteins was affected. Fluorescence microscopy and cell lysate immunoprecipitation studies revealed that apoA-V is not associated with apoB intracellularly, yet immunoprecipitation of apoA-V from the cell culture medium resulted in coprecipitation of apoB. These data suggest that the apoA-V association with apoB-containing lipoproteins is a postsecretory event. Confocal fluorescence microscopy revealed the presence of apoA-V in distinct cellular structures. Based on Nile Red staining, we identified these structures to be intracellular lipid droplets. These data suggest that apoA-V has a unique association with cellular lipids and, therefore, may be involved in the storage or mobilization of intracellular lipids.

Lipid Homeostasis and Lipoprotein Secretion in Niemann-Pick C1-deficient Hepatocytes

Niemann-Pick C (NPC) disease is a fatal inherited disorder characterized by an accumulation of cholesterol and other lipids in late endosomes/lysosomes. Although this disease is considered to be primarily a neurodegenerative disorder, many NPC patients suffer from liver disease. We have investigated alterations that occur in hepatic lipid homeostasis using primary hepatocytes isolated from NPC1-deficient mice. The cholesterol content of Npc1(-/-) hepatocytes was 5-fold higher than that of Npc1(+/+) hepatocytes; phospholipids and cholesteryl esters also accumulated. In contrast, the triacylglycerol content of Npc1(-/-) hepatocytes was 50% lower than of Npc1(+/+) hepatocytes. We hypothesized that the cholesterol sequestration induced by NPC1 deficiency might inhibit very low density lipoprotein secretion. However, this process was enhanced by NPC1 deficiency and the secreted particles were enriched in cholesteryl esters. We investigated the mechanisms responsible for these changes. The synthesis of phosphatidylcholine, cholesteryl esters, and cholesterol in hepatocytes was increased by NPC1 deficiency and the amount of the mature form of sterol response element-binding protein-1 was also increased. These observations indicate that the enhanced secretion of lipoproteins from NPC1-deficient hepatocytes is due, at least in part, to increased lipid synthesis.

Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-plasmodium toxicity

We previously showed that the in vitro intraerythrocytic development of the malarial agent Plasmodium falciparum is strongly inhibited by secreted phospholipases A(2) (sPLA(2)s) from animal venoms. Inhibition is dependent on enzymatic activity and requires the presence of serum lipoproteins in the parasite culture medium. To evaluate the potential involvement of host lipoproteins and sPLA(2)s in malaria, we investigated the interactions between bee venom phospholipase A(2) (bvPLA(2)), human triglyceride-rich lipoproteins, and infected erythrocytes. Even at high enzyme concentration (100x IC(50)), bvPLA(2) binding to Plasmodium-infected or normal erythrocytes was not detected. On the contrary, tight association with lipoproteins was observed through the formation of buoyant bvPLA(2)/lipoprotein complexes. Direct involvement of the hydrolysis lipid products in toxicity was demonstrated. Arachidonic acid (C20:4), linoleic acid (C18:2), and, to a lesser extent, docosahexaenoic acid (C22:6) appeared as the main actors in toxicity. Minimal oxidation of lipoproteins enhanced toxicity of the lipolyzed particles and induced their interaction with infected or normal erythrocytes. Fresh or oxidized lipolyzed lipoproteins induced the parasite degeneration without host cell membrane disruption, ruling out a possible membranolytic action of fatty acids or peroxidation products in the death process. In conclusion, our data enlighten on the capability of secreted PLA(2)s to exert cytotoxicity via the extracellular generation of toxic lipids, and raise the question of whether such mechanisms could be at play in pathophysiological situations such as malaria.

A Long-Term Study of Dyslipidemia and Dyslipoproteinemia in Stable Post-Renal Transplant Patients

Background. Dyslipidemia is a major risk factor for atherosclerotic disease in renal transplant patients. Methods. The serum levels of lipids and lipoproteins were determined in the same 12 post-renal transplant patients (TX) 10–29 and 73–122 months after transplantation. Thirteen healthy subjects—i.e., without diabetes, endocrine disease, liver disease, active inflammatory disease, glucose intolerance, malignancy, obesity, and urinary protein—were used as a reference group. TX patients had stable renal function. Twelve patients received cyclosporine A and prednisone, six received lovastatin, and one received rapa and prednisone. Lipids and lipoprotein (apo)AI and B were determined using Roche kits. An anti-apoB antibody was used to separate apoB-containing apoCIII and apoE lipoproteins as triglyceride-rich lipoproteins (TRLs) in the non-HDL fraction from apoCIIInonB and apoEnonB in the HDL fraction. Results. In both groups of post-renal transplant patients, a statistically significant increase of TG, TC, and non-HDL-C levels was observed. Moreover, statistically significant changes were shown in total apoCIII and apoCIIInonB, as well as in TG/HDL-C and apoAI/apoCIII ratios, as compared to the reference group. On the other hand, in TX patients 73–122 months after transplantation, significantly higher concentrations of TC, LDL-C, and especially non-HDL-C were observed. It was shown that apoCIII, apoCIIInonB, apoB:CIII, and lipid and lipoprotein ratios as risk factors of atherosclerosis and renal risk factors were higher in these patients 73–122 months after transplantation. Conclusion. TX patients in a long-term study showed that they had disturbed lipoprotein composition, and its consequence was hyperlipidemia, perhaps partly due to the increased use of immunosuppressants and steroids.

Disturbed lipoprotein composition in non-dialyzed, hemodialysis, continuous ambulatory peritoneal dialysis and post-transplant patients with chronic renal failure

Studies were carried out in 183 non-dialyzed, 123 hemodialysis, 81 continuous ambulatory peritoneal dialysis and 35 post-transplant patients and in 103 healthy subjects as a reference group. Lipids and apolipoprotein (apo)AI and apoB were determined using Roche kits. An anti-apoB antibody was used to separate apoB-containing apoCIII and apoE-triglyceride-rich lipoprotein (TRL) in the non-high-density lipoprotein (non-HDL) fraction from apoCIIInonB and apoEnonB in the HDL fraction in four groups of patients with chronic renal failure (CRF) and healthy subjects. Multivariate linear regression analysis was used to investigate the relationship between triglyceride (TG) or HDL-cholesterol (HDL-C) concentrations and lipoproteins. Dyslipidemia varied according to the degree of renal insufficiency, the type of dialysis and therapy regime in CRF patients. Lipoprotein disturbances were manifested by increased TG, non-HDL-C and TRL concentrations, and decreased HDL-C and apoAI concentrations, whereas post-renal transplant patients showed normalization of lipid and lipoprotein profiles, except for TG levels and total apoCIII and apoCIIInonB. The present study indicates that CRF patients have disturbed lipoprotein composition, and that hypertriglyceridemia and low HDL-C concentrations in these patients are multifactorial, being secondary to disturbed lipoproteins. The method using anti-apoB antibodies to separate apoB-containing lipoproteins in the non-HDL fraction from non-apoB-containing lipoproteins in HDL can be used in the diagnosis and treatment of patients with progression of renal failure or atherosclerosis. The variability of TG and HDL-C concentrations depends on the variability of TRL and cholesterol-rich lipoprotein concentrations, but the decreases in TG and increases in HDL-C concentrations are caused by apoAI concentration variability. These relationships, however, need to be confirmed in further studies.

The Possible Origin of Oxidized Low-Density Lipoproteins in the Circulation

Oxidized low-density lipoprotein (OxLDL) is thought to be involved in the early development of atherosclerosis through foam cell formation. However, the actual nature of OxLDL found in vivo remained obscure until recently. We have established a sensitive method of measuring OxLDL present in human plasma by utilizing an anti-OxLDL monoclonal antibody, DLH3, together with an anti-apoB antibody. OxLDL levels appeared to increase in patients with acute myocardial infarction, cerebral infarction and several other pathological conditions. Evidence is accumulating which supports the close relation between vascular diseases and plasma OxLDL levels. In this article, a major concern is the source of the OxLDL. Our recent studies on the OxLDL present in both atherosclerotic lesions and in plasma, together with observations of minimally modified LDL, provide some further clues to the understanding of some of the features and behavior of OxLDL in the circulation. A possible scenario detailing the origin and dynamics of OxLDL in vivo is discussed.

Atherosclerosis and Sensitive Determination of Oxidized LDL Using Monoclonal Antibody

AbstractFor Abstract see ChemInform Abstract in Full Text.

Oxidized low-density lipoproteins: what is understood and what remains to be clarified.

Oxidized low-density lipoprotein (OxLDL) has previously been thought to promote atherogenesis through foam cell formation. However, the actual nature of OxLDL present in vivo remained obscure until recently. We have produced a monoclonal antibody, DLH3, which specifically binds to OxLDL but not to native LDL. The presence of OxLDL in the LDL fraction of human plasma was demonstrated by introducing a sandwich ELISA procedure using DLH3 together with an anti-apoB antibody. Furthermore, OxLDL levels appeared to increase in certain pathological conditions including acute myocardial infarction and carotid artery atherosclerosis. Accumulation of OxLDL in atherosclerotic lesions has also been demonstrated by immunohistochemical and biochemical studies using the DLH3 antibody. This antibody recognizes oxidized phosphatidylcholines (OxPC) generated during oxidative modification of LDL, and OxPC-apoB adducts formed in OxLDL are the presumed antigens. Measuring OxLDL in plasma would be a useful diagnostic tool for cardiovascular diseases. However, there still remain some major questions related to OxLDL, the answers to which are crucial for understanding the mechanisms of atherogenesis.

Atherosclerosis and sensitive determination of oxidized LDL using monoclonal antibody

Oxidized low-density lipoprotein (OxLDL) is thought to be involved in atherosclerotic lesion formation. We established a monoclonal antibody, DLH3, that recognizes oxidized phosphatidylcholine (OxPC) formed in OxLDL. A sensitive method for detecting OxLDL was enabled by a sandwich ELISA procedure utilizing DLH3 together with an anti-apoB antibody. Using the method, we demonstrated the presence of OxLDL in human circulating plasma for the first time, and the plasma OxLDL level in healthy subjects was estimated to be about 0.1 ng/microgram LDL protein. OxLDL levels in patients with acute myocardial infarction are more than 3 times higher than in controls. Thus the plasma OxLDL level could be a good marker for cardiovascular disease. There are multiple metabolic enzymes for OxPC in plasma. We demonstrated that lecithin: cholesterol acyltransferase (LCAT) is capable of metabolizing OxPC molecules in OxLDL, and that the plasma OxLDL level in patients with familial LCAT deficiency was about 3.5 times higher than in controls. OxLDL in vivo is likely to be metabolized by enzymatic activities in plasma, the reticuloendothelial system including Kupffer cells, and immunological responses. The OxLDL levels determined by this analytical procedure would reflect the physiologic balance between oxidative modification of LDL and metabolic clearance of OxLDL.

Accumulation of LOX-1 Ligand in Plasma and Atherosclerotic Lesions of Watanabe Heritable Hyperlipidemic Rabbits: Identification by a Novel Enzyme Immunoassay

LOX-1 (lectin-like oxidized LDL receptor) is a newly identified cell surface receptor for oxidized LDL mainly expressed in endothelial cells. Recombinant soluble LOX-1(LOX-Fc) was generated by fusing the extracellular domain of LOX-1 with the Fc portion of IgG. A novel sandwich enzyme immunoassay specific for LOX-1 ligand is designed, using LOX-Fc and anti-apoB antibody. This immunoassay was used to determine LOX-1 ligand activity in normal and Watanabe heritable hyperlipidemic (WHHL) rabbit plasma. LOX-Fc was further applied for staining of atherosclerotic lesions of WHHL rabbits. LOX-1 ligand levels were significantly elevated in the plasma of hyperlipidemic rabbits compared with controls. Furthermore, LOX-1 ligand activity was detected in the atherosclerotic lesions in situ. These results support the potential roles of LOX-1 interacting with its ligand in the pathogenesis of atherosclerosis, which is enhanced in hyperlipidemia.

Sphingomyelinase converts lipoproteins from apolipoprotein E knockout mice into potent inducers of macrophage foam cell formation.

The apoE knockout (E0) mouse is one of the most widely used animal models of atherosclerosis, and there may be similarities to chylomicron remnant-induced atherosclerosis in humans. Although the lesions of these mice contain large numbers of cholesteryl ester (CE)-laden macrophages (foam cells), E0 plasma lipoproteins are relatively weak inducers of cholesterol esterification in macrophages. Previous in vivo work has suggested that arterial wall sphingomyelinase (SMase) may promote atherogenesis in the E0 mouse, perhaps by inducing subendothelial lipoprotein aggregation and subsequent foam cell formation. The goal of the present study was to test the hypothesis that the modification of E0 lipoproteins by SMase converts these lipoproteins into potent inducers of macrophage foam cell formation. When d<1.063 E0 lipoproteins were pretreated with SMase and then incubated with E0 macrophages, cellular CE mass and stimulation of the cholesterol esterification pathway were increased approximately 5-fold compared with untreated lipoproteins. SMase-treated E0 lipoproteins were more potent stimulators of cholesterol esterification than either E0 lipoproteins in the presence of lipoprotein lipases or oxidized E0 lipoproteins. The uptake and degradation of SMase-treated E0 lipoproteins by macrophages were saturable and specific and substantially inhibited by partial proteolysis of cell-surface proteins. Uptake and degradation were diminished by an anti-apoB antibody and by competition with human S(f) 100-400 hypertriglyceridemic VLDL, raising the possibility that a receptor that recognizes apoB-48 might be involved. In conclusion, SMase-modification of E0 lipoproteins, a process previously shown to occur in lesions, may be an important mechanism for foam cell formation in this widely studied model of atherosclerosis. Moreover, the findings in this report may provide important clues regarding the atherogenicity of chylomicron remnants in humans.