Presecretory degradation of apolipoprotein [a] is mediated by the proteasome pathway

Plasma levels of atherogenic lipoprotein [a] (Lp[a]) vary over a 1000-fold range and are largely determined by the gene for its unique glycoprotein, apolipoprotein [a] (apo[a]). The apo[a] locus comprises more than 100 alleles, encoding proteins from <300 to >800 kDa. Using primary baboon hepatocyte cultures, we previously demonstrated that differences in the secretion efficiency of apo[a] allelic variants contribute to the variation in plasma Lp[a] levels. In the current study, we investigated the mechanism of apo[a] presecretory degradation. The proteasome inhibitors, acetyl-leucyl-leucyl-norleucinal and lactacystin, prevented apo[a] degradation and increased apo[a] secretion. Transfection with an HA-tagged ubiquitin construct demonstrated the accumulation of ubiquitinated apo[a] in the presence of lactacystin. These results suggest a role for the cytoplasmic proteasome in apo[a] proteolysis. Apo[a] that accumulated intracellularly in the presence of lactacystin remained sensitive to endo-B-N-glucosaminidase H, and apo[a] degradation was reversibly inhibited by brefeldin A, suggesting that transport to a post-endoplasmic reticulum (ER) pre-medial Golgi compartment is required for apo[a] degradation. Newly synthesized apo[a] bound to the ER chaperone calnexin and conditions that enhanced this interaction prevented apo[a] degradation, suggesting that calnexin can protect apo[a] from proteolysis. These studies provide further support for the role of the proteasome in endoplasmic reticulum quality control, and expand this role to one that influences plasma levels of the atherogenic lipoprotein Lp[a].-White, A. L., B. Guerra, J. Wang, and R. E. Lanford. Presecretory degradation of apolipoprotein[a] is mediated by the proteasome pathway.     

Measurement of oxidation in plasma Lp(a) in CAPD patients using a novel ELISA

BACKGROUND: LGE2 is produced by the cyclooxygenase- or free radical-mediated modification of arachidonate and is formed during the oxidation of low density lipoprotein (LDL) with subsequent adduction to lysine residues in apo B. We have developed a sensitive enzyme-linked sandwich immunosorbent assay (ELISA) for detection and measurement of LGE2-protein adducts as an estimate of oxidation of plasma LDL and Lp(a). METHODS: The assay employs rabbit polyclonal antibodies directed against LGE2-protein adducts that form pyrroles, and alkaline phosphatase-conjugated polyclonal antibodies specific for apo B or apo (a). It demonstrates a high degree of specificity, sensitivity and validity. RESULTS: Epitopes characteristic for LGE2-pyrroles were quantified in patients with end-stage renal disease (ESRD) that had undergone continuous ambulatory peritoneal dialysis (CAPD) and in a gender- and age-matched control population. In addition to finding that both LDL and Lp(a) levels were elevated in CAPD patients, we also found that plasma Lp(a) but not LDL was more oxidized in CAPD patients when compared to corresponding lipoproteins from healthy subjects. Using density gradient ultra-centrifugation of plasma samples, we found that modified Lp(a) floats at the same density as total Lp(a). CONCLUSIONS: The results of this study demonstrate that oxidation of plasma Lp(a) is a characteristic of ESRD patients undergoing CAPD. This ELISA may be useful for further investigations on oxidation of lipoproteins in the circulation of specific patient populations.     

Inhibition of N-linked glycosylation results in retention of intracellular apo[a] in hepatoma cells, although nonglycosylated and immature forms of apolipoprotein[a] are competent to associate with apolipoprotein B-100 in vitro

Apolipoprotein[a] (apo[a]) is a highly polymorphic glycoprotein that forms a covalent complex with apolipoprotein B-100 (apoB-100), producing a lipoprotein species referred to as lipoprotein[a] (Lp[a]). We have studied the effects of alterations in glycosylation of apo[a] on its intracellular processing and secretion as well as its ability to associate with low density lipoprotein (LDL) apoB-100. HepG2 cells transfected with a 6 kringle IV (6 K-IV) apo[a] minigene were treated with tunicamycin, an inhibitor of N-linked glycosylation, which eliminated apo[a]-B-100 complexes from the media. Tunicamycin treatment also reduced secretion of the 6 K-IV apo[a] protein from transfected McA-RH7777 cells by approximately 50%, but completely eliminated secretion of apo[a] species containing 9 and 17 K-IV repeats. Mixing experiments, performed with radiolabeled media (+/-tunicamycin) from transfected McA-RH7777 cells, demonstrated no alteration in the extent of association of apo[a] with human LDL. Similar mixing experiments using culture media from glycosylation-defective mutant chinese hamster ovary (CHO) cells transfected with the same apo[a] minigene showed identical results. Apo[a] secretion was demonstrated in all mutant cell lines in the absence of either N- or O-linked (or both) glycosylation. The mechanisms underlying the reduced secretion of apo[a] from transfected hepatoma cells were examined by pulse-chase radiolabeling and apo[a] immunoprecipitation. Tunicamycin treatment altered the efficiency of precursor apo[a] processing from the ER by increasing its ER retention time. The increased accumulation of precursor apo[a] in the ER was associated with alterations in the kinetics of association with two resident endoplasmic reticulum (ER) chaperone proteins, calnexin and BiP. These findings suggest that the glycosylation state and size of apo[a] appear to play a role in regulating its efficient exit from the endoplasmic reticulum. However, neither N- nor O-linked glycosylation of apo[a] exerts a major regulatory role in its covalent association with apoB-100.     

Neomycin inhibits secretion of apolipoprotein[a] by increasing retention on the hepatocyte cell surface

Neomycin therapy reduces plasma levels of low density lipoprotein and lipoprotein[a] (Lp[a]). To determine whether neomycin directly alters the biogenesis of Lp[a], we have examined the effect of neomycin on apolipoprotein[a] (apo[a]) synthesis and secretion in primary cultures of baboon hepatocytes. Using this system, we have previously shown that apo[a] is synthesized as a lower molecular weight precursor that upon maturation becomes associated with the cell surface before release into the culture medium. Treatment of hepatocytes with 10 mM neomycin reduced levels of apo[a] in the culture medium by as much as 12-fold. Although a portion of the reduced secretion could be accounted for by a reduction in total protein synthesis, the greatest effect of neomycin on apo[a] secretion was to decrease the release of mature apo[a] from the hepatocyte cell surface into the culture medium. Treatment of hepatocyte cultures with trypsin confirmed that mature apo[a] in neomycin-treated cells was still transported to the cell surface. Examination of related antibiotics demonstrated that inhibition of apo[a] secretion is a general property shared by the deoxystreptamine antibiotics. The mechanism by which neomycin affects the apo[a]-cell surface interaction is not known, but neomycin is known to perturb cell surface membranes, inhibit the interaction of some ligands with their cell surface receptors, and inhibit the metabolism of phosphatidylinositol 4,5 biphosphate. These studies suggest that cell surface association of apo[a] may play a role in Lp[a] biogenesis in vivo.     

Effects of apoE gene polymorphism on Lp(a) concentrations depend on the size of apo(a): a study of 466 white men

Polymorphisms in the genes for the low-density lipoprotein (LDL) receptor ligands, apolipoprotein E (apoE), and apolipoprotein B (apoB) are associated with variation in plasma levels of LDL cholesterol. Lp(a) lipoprotein(a) [Lp(a)] is LDL in which apoB is attached to a glycoprotein called apolipoprotein(a) [apo(a)]. Apo(a) has several genetically determined isoforms differing in molecular weight, which are inversely correlated with Lp(a) concentrations in blood. The interaction of apo(a) with triglyceride-rich lipoproteins differs with the size of apo(a), and therefore the effects of apoE gene polymorphism on Lp(a) levels could also depend on apo(a) size. We have investigated the possible effect of genetic variation in the apoE and apoB genes on plasma Lp(a) concentrations in 466 white men with different apo(a) phenotypes. Overall there was no significant association between the common apoE polymorphism and Lp(a), but in the subgroup with apo(a)-S4, concentrations of Lp(a) differed significantly among the apoE genotypes (P = 0.05). Lp(a) was highest in the apoE genotypes epsilon 2 epsilon 3 and epsilon 3 epsilon 3 and lowest in genotype epsilon 3 epsilon 4, and the apoE polymorphism was estimated to account for about 2.4% of the variation in Lp(a). In contrast, in the subgroup with apo(a)-S2 Lp(a) was significantly lower (P = 0.04) in apoE genotype epsilon 2 epsilon 3 than in genotype epsilon 3 epsilon 3. Lp(a) concentrations did not differ among the XbaI (P = 0.65) or SP 24/27 (P = 0.26) polymorphisms of the apoB gene. The expected effects of both apoE and apoB polymorphism on LDL levels were significant in the whole population sample and in subjects with large-sized apo(a) isoforms (P < 0.01), whereas no effect was seen in those with low molecular weight apo(a) isoforms. We conclude that the influence of apoE genotypes on Lp(a) concentrations depends on the size of the apo(a) molecule in Lp(a), possibly because both apo(a)-S4 and apoE4 have high affinity for triglyceride-rich lipoproteins and may be taken up and degraded rapidly by remnant receptors.     

Polymorphism of apolipoprotein E, lipoprotein(a), and other lipoproteins in children with type I diabetes

We assessed the effect of particular apolipoprotein (apo) E phenotypes, lipoprotein(a) [Lp(a)], and other lipoproteins on the development of dyslipoproteinemia in 450 patients with type I diabetes, ages 13-14 years. The control group consisted of 450 healthy school children of both sexes, ages 13-14 years. Both groups were found to be normolipidemic, but the concentration of Lp(a) was significantly (P < 0.05) higher in the diabetic children than in the control group. Apo E 3/2 and apo E 4/4 phenotypes were more frequent in the group of diabetics. Diabetics with the apo E 3/3 phenotype had higher concentrations of very-low-density lipoprotein (VLDL) and Lp(a), and lower concentrations of low-density lipoprotein (LDL) than the apo E 3/3 nondiabetics. For apo E 3/2 phenotypes, total cholesterol, LDL cholesterol, LDL, apo A-I, and Lp(a) concentrations were higher in the diabetic children than in the control group; for apo E 4/3 phenotypes, this was true for triglycerides and VLDL cholesterol. The distribution of Lp(a) lipoprotein concentrations between 0.01 and > 0.5 g/L indicated a more frequent occurrence of higher Lp(a) values in diabetic children than in the control group. Results of this study indicate that an increased concentration of Lp(a) lipoprotein and apo E 3/2 and apo E 4/3 phenotypes contribute to the expression of dyslipoproteinemia in type I diabetes in childhood.     

Binding of the G protein betagamma subunit to multiple regions of G protein-gated inward-rectifying K+ channels

Lipoprotein-(a) [Lp(a)] is a highly atherogenic lipoprotein with unknown function, consisting of a low-density lipoprotein (LDL) core and the apo(a) glycoprotein. The characteristic structural feature of apo(a) is the presence of multiple so called "kringle' repeats which are in part identical and in part exhibit slight sequence differences. The assembly of apo(a) and LDL, which is determinant for plasma Lp(a) levels, takes place extracellularly and requires specific structural motifs in apo(a) and apoB. Here we studied the structural features in apo(a) necessary for high-efficient assembly. Thirteen recombinant apo(a) glycoproteins, which differed in the set of kringle-IV (K-IV) motifs, were expressed in COS-7 cells and incubated with LDL. The rate of total and disulfide-stabilized Lp(a) complex formation was measured by an immunochemical assay. Constructs containing K-IV T(type)5-T10 yielded almost 100% total and 80% stable complexes, respectively. Deletion or replacement of the different kringles revealed that K-IV T6 and T7 were responsible for the high-yield assembly and that K-IV T5 had an amplifying effect. Increasing the absolute number of K-IV repeats had an additional amplifying effect. The rate of Lp(a) assembly correlated strongly with the affinity of these constructs to Lys-Sepharose. Our results have implications for understanding the metabolism of Lp(a) and may help to design strategies for searching natural apo(a) mutants with aberrant plasma Lp(a) levels.     

LDL-unbound apolipoprotein(a) and carotid atherosclerosis in hemodialysis patients

High lipoprotein(a) [Lp(a)] plasma concentrations, which are genetically determined by apo(a) size polymorphism, are directly associated with an increased risk for atherosclerosis. Patients with end-stage renal disease (ESRD), who show an enormous prevalence of cardiovascular disease, have elevated plasma concentrations of Lp(a). In recent studies we were able to show that apo(a) size polymorphism is a better predictor for carotid atherosclerosis and coronary artery disease in hemodialysis patients than concentrations of Lp(a) and other lipoproteins. Less than 5% of apo(a) in plasma exists in a low-density lipoprotein (LDL)-unbound form. This "free" apo(a) consists mainly of disintegrated apo(a) molecules of different molecular weight, ranging from about 125 to 360 kDa. LDL-unbound apo(a) molecules are elevated in patients with ESRD. The aim of this study was therefore to investigate whether the LDL-unbound form of apo(a) contributes to the prediction of carotid atherosclerosis in a group of 153 hemodialysis patients. The absolute amount of LDL-unbound apo(a) showed a trend to increasing values with the degree of carotid atherosclerosis, but the correlation of Lp(a) plasma concentrations with atherosclerosis was more pronounced. In multivariate analysis the two variables were related to neither the presence nor the degree of atherosclerosis. Instead, the apo(a) phenotype took the place of Lp(a) and LDL-unbound apo(a). After adjustment for other variables, the odds ratio for carotid atherosclerosis in patients with a low molecular weight apo(a) phenotype was about 5 (p<0.01). This indicates a strong association between the apo(a) phenotype and the prevalence of carotid atherosclerosis. Finally, multivariate regression analysis revealed age, angina pectoris and the apo(a) phenotype as the only significant predictors of the degree of atherosclerosis in these patients. In summary, it seems that LDL-unbound apo(a) levels do not contribute to the prediction of carotid atherosclerosis in hemodialysis patients. However, this does not mean that "free", mainly disintegrated, apo(a) has no atherogenic potential.     

Lipoprotein(a) in health and disease

Lipoprotein(a) [Lp(a)] represents an LDL-like particle to which the Lp(a)-specific apolipoprotein(a) is linked via a disulfide bridge. It has gained considerable interest as a genetically determined risk factor for atherosclerotic vascular disease. Several studies have described a correlation between elevated Lp(a) plasma levels and coronary heart disease, stroke, and peripheral atherosclerosis. In healthy individuals, Lp(a) plasma concentrations are almost exclusively controlled by the apo(a) gene locus on chromosome 6q2.6-q2.7. More than 30 alleles at this highly polymorphic gene locus determine a size polymorphism of apo(a). There exists an inverse correlation between the size (molecular weight) of apo(a) isoforms and Lp(a) plasma concentrations. The standardization of Lp(a) quantification is still an unresolved task due to the large particle size of Lp(a), the presence of two different apoproteins [apoB and apo(a)], and the large size polymorphism of apo(a) and its homology with plasminogen. A working group sponsored by the IFCC is currently establishing a stable reference standard for Lp(a) as well as a reference method for quantitative analysis. Aside from genetic reasons, abnormal Lp(a) plasma concentrations are observed as secondary to various diseases. Lp(a) plasma levels are elevated over controls in patients with nephrotic syndrome and patients with end-stage renal disease. Following renal transplantation, Lp(a) concentrations decrease to values observed in controls matched for apo(a) type. Controversial data on Lp(a) in diabetes mellitus result mainly from insufficient sample sizes of numerous studies. Large studies and those including apo(a) phenotype analysis came to the conclusion that Lp(a) levels are not or only moderately elevated in insulin-dependent patients. In noninsulin-dependent diabetics, Lp(a) is not elevated. Conflicting data also exist from studies in patients with familial hypercholesterolemia. Several case-control studies reported elevated Lp(a) levels in those patients, suggesting a role of the LDL-receptor pathway for degradation of Lp(a). However, recent turnover studies rejected that concept. Moreover, family studies also revealed data arguing against an influence of the LDL receptor for Lp(a) concentrations. Several rare diseases or disorders, such as LCAT- and LPL-deficiency as well as liver diseases, are associated with low plasma levels or lack of Lp(a).     

Sib-pair analysis detects elevated Lp(a) levels and large variation of Lp(a) concentration in subjects with familial defective ApoB

Whether or not Lp(a) plasma levels are affected by the apoB R3500Q mutation, which causes Familial Defective apoB (FDB), is still a matter of debate. We have analyzed 300 family members of 13 unrelated Dutch index patients for the apoB mutation and the apolipoprotein(a) [apo(a)] genotype. Total cholesterol, LDL-cholesterol, and lipoprotein(a) [Lp(a)] concentrations were determined in 85 FDB heterozygotes and 106 non-FDB relatives. Mean LDL levels were significantly elevated in FDB subjects compared to non-FDB relatives (P < 0.001). Median Lp(a) levels were not different between FDB subjects and their non-FDB relatives. In contrast, sib-pair analysis demonstrated a significant effect of the FDB status on Lp(a) levels. In sib pairs identical by descent for apo(a) alleles but discordant for the FDB mutation (n = 11) each sib with FDB had a higher Lp(a) level than the corresponding non-FDB sib. Further, all possible sib pairs (n = 105) were grouped into three categories according to the absence/presence of the apoB R3500Q mutation in one or both subjects of a sib pair. The variability of differences in Lp(a) levels within the sib pairs increased with the number (0, 1, and 2) of FDB subjects present in the sib pair. This suggests that the FDB status increases Lp(a) level and variability, and that apoB may be a variability gene for Lp(a) levels in plasma.     

Low density lipoprotein receptor-negative mice expressing human apolipoprotein B-100 develop complex atherosclerotic lesions on a chow diet: no accentuation by apolipoprotein(a)

We have generated mice with markedly elevated plasma levels of human low density lipoprotein (LDL) and reduced plasma levels of high density lipoprotein. These mice have no functional LDL receptors [LDLR-/-] and express a human apolipoprotein B-100 (apoB) transgene [Tg(apoB+/+)] with or without an apo(a) transgene [Tg(apoa+/-)]. Twenty animals (10 males and 10 females) of each of the following four genotypes were maintained on a chow diet: (i) LDLR-/-, (ii) LDLR-/-;Tg(apoa+/-), (iii) LDLR-/-;Tg(apoB+/+), and (iv)LDLR-/-;Tg(apoB+/+);Tg(apo+/-). The mice were killed at 6 mo, and the percent area of the aortic intimal surface that stained positive for neutral lipid was quantified. Mean percent areas of lipid staining were not significantly different between the LDLR-/- and LDLR-/-;Tg(apoa+/-) mice (1.0 +/- 0.2% vs. 1.4 +/- 0.3%). However, the LDLR-/-;Tg(apoB+/+) mice had approximately 15-fold greater mean lesion area than the LDLR-/- mice. No significant difference was found in percent lesion area in the LDLR-/-;Tg(apoB+/+) mice whether or not they expressed apo(a) [18.5 +/- 2.5%, without lipoprotein(a), Lp(a), vs. 16.0 +/- 1.7%, with Lp(a)]. Histochemical analyses of the sections from the proximal aorta of LDLR-/-;Tg(apoB+/+) mice revealed large, complex, lipid-laden atherosclerotic lesions that stained intensely with human apoB-100 antibodies. In mice expressing Lp(a), large amounts of apo(a) protein colocalized with apoB-100 in the lesions. We conclude that LDLR-/-; Tg(apoB+/+) mice exhibit accelerated atherosclerosis on a chow diet and thus provide an excellent animal model in which to study atherosclerosis. We found no evidence that apo(a) increased atherosclerosis in this animal model.     

Elevated lipoprotein (a) levels in primary nephrotic syndrome

Elevated plasma levels of total cholesterol and increase in the hepatic synthesis of some apo B-containing lipoproteins have been noted in the nephrotic syndrome. Apoprotein (a), the apolipoprotein distinguishing lipoprotein (a) [Lp(a)] from low-density lipoprotein, is equally of hepatic origin, and Lp(a) recently has been shown to possess both atherogenic and thrombogenic activities. However, little is known of Lp(a) levels in nephrotic patients. We measured plasma Lp(a) concentrations in 11 patients with primary nephrotic syndrome in the absence of hematuria, hypertension, and renal insufficiency. Histologic lesions were minimal-change disease in five cases, membranous glomerulopathy in four cases, and focal and segmental glomerulosclerosis in two cases. Mean levels of Lp(a) (98 +/- 92 mg/dL [mean +/- SD]) were markedly elevated in the nephrotic patients as compared with the controls (14 +/- 13 mg/dL). No correlation was noted between plasma Lp(a) and proteinuria, albuminemia, total cholesterolemia, low-density lipoprotein cholesterol, apoprotein B100, or plasminogen. Furthermore, there was no correlation between Lp(a) levels and apoprotein (a) isoform size. In four patients, the level of Lp(a) decreased approximately fourfold after remission of the nephrotic syndrome under corticosteroid treatment. Our observation that Lp(a) levels are elevated in the nephrotic syndrome is consistent with the hypothesis that these patients may be at an increased risk of cardiovascular and thrombotic complications.     

Differences in Lp[a] concentrations and apo[a] polymorphs between black and white Americans

Lp[a] concentrations in nmol/L and apo[a] size isoforms, expressed in terms of the relative number of apo[a] kringle 4 (K4) repeats, were determined in 3959 whites and blacks from four U.S. communities. Plasma Lp[a] analyses were performed by an ELISA method insensitive to apo[a] size heterogeneity and apo[a] size isoforms were determined by high resolution agarose gel electrophoresis. Allele frequencies were estimated by maximum likelihood methods in order to account for the presence of null alleles and coalescence of hands on gels. The apo[a] allele frequencies and phenotype distributions differed significantly between blacks and whites (P < 0.0001). Blacks had a higher relative frequency of the intermediate alleles K4(22) through K4(28) whereas whites had a higher relative frequency of the small alleles K4(17) through K4(24) and large alleles K4(29) through K4(33). The estimated frequency of the null allele was low in both blacks (1.0%) and whites (6.7%). The Lp[a] distribution was less skewed and Lp[a] concentrations were higher in blacks than whites (mean 94 nmol/L and 48 nmol/L, median 74 nmol/L and 20 nmol/L for blacks and whites, respectively). The relationship between apo[a] size and Lp[a] concentration also differed significantly between these two racial groups. For the large polymorphs (> 31 K4 repeats) both blacks and whites exhibited uniformly low Lp[a] values. For the intermediate isoforms K4(20) through K4(30), a considerable range of Lp[a] values was evident in blacks; the median Lp[a] for each isoform increased nearly linearly as the apo[a] size decreased. In contrast in whites there was little change in median Lp[a] concentrations for isoforms K4(20) through K4(30). For the small apo[a] size (< 20 K4) both blacks and whites exhibited high median Lp[a] levels and a wide variation of Lp[a] levels. The major difference in Lp[a] levels between the two racial groups occurred in the intermediate size isoform range of K4(20) through K4(25). In conclusion, whites and blacks differ significantly in Lp[a] concentrations, allele and phenotype frequencies, and in the relationship between apo[a] size isoform and Lp[a] concentration.     

Apolipoprotein(a) binds to low-density lipoprotein at two distant sites in lipoprotein(a)

Lipoprotein(a) [Lp(a)] consists of low-density lipoprotein (LDL) and apolipoprotein(a) [apo(a)] linked with a disulfide bond. Scanning force microscopy (SFM) of Lp(a) showed, for the first time, a belt-like structure of apo(a) with both ends attached to a spherical LDL. The two ends of apo(a) were bound to the LDL sphere at two distant sites. Occasionally, the ends were attached to two touching spheres. Under the same imaging conditions, LDL appeared as individual spheres. Electron microscopy (EM) studies of Lp(a) by several groups over the past decade failed to reveal this belt-like structure of apo(a). Images of isolated apo(a) in air or in phosphate buffer showed apo(a) as individual belts, and these belts tended to crowd together. Lp(a) formed leaf-like aggregates; apo(a) aggregates were fishnet-like, whereas LDL aggregates were less characteristic. Quantitative analysis of Lp(a) showed the diameter of the LDL to be 24.8 +/- 8.7 nm (n = 46), which is close to the reported value of 24.2 +/- 4.2 nm found with EM. The length of the belts attached to the spheres was measured to be 173.5 +/- 6.6 nm (n = 15). I also found, by using a functionalized tip, that the interaction force between apo(a) and its ligand, lysine, was related to the ionic strength of the bulk solution. This force can be reduced by the presence of epsilon-aminocaproic acid.     

Responses of dorsal column nuclei neurons in rats with experimental mononeuropathy

During nerve cell degeneration, cholesterol released from the degrading cells is conserved through the formation of a cholesterol-apolipoprotein (apo) E complex which is subsequently taken up by regenerating nerve cells. The aim of the present project was to identify the physiologically relevant lipoprotein receptor for this lipoprotein complex which has remained elusive. HDL was separated into apo E-rich and apo E-poor subfractions and labelled with [14C]-sucrose. Labelled apo E-rich HDL bound to rat brain membranes in a time- and ligand concentration-dependent manner and was a saturable process. Essentially no binding occurred with [14C]-apo E-poor HDL or with free apo E. Binding was partially inhibited by low density lipoprotein (LDL) and by alpha 2-macroglobulin. These results provide new evidence that native apoE-rich HDL particles resembling those present in the brain bind to rat brain membranes and that the binding may be due, at least in part, to the LDL receptor and to the LDL-receptor related protein. Evidence was also provided for the presence of a receptor which binds [14C]-sucrose human apoE-rich HDL in human brain. Characterisation of the receptor which mediates the uptake of cholesterol from HDL-like complexes by brain cells is important in understanding the role of apoE in the central nervous system and of the alterations which occur in disorders such as Alzheimer's disease.     

Lipoprotein lipase increases lipoprotein binding to the artery wall and increases endothelial layer permeability by formation of lipolysis products

Mechanisms responsible for the accumulation of low-density lipoprotein (LDL) were investigated in a new model, the perfused hamster aorta. To do this, we developed a method to study LDL flux in real time in individually perfused arteries; each artery served as its own control. Using quantitative fluorescence microscopy, the rates of LDL accumulation and efflux were separately determined. Perfusion of arteries with buffer plus lipoprotein lipase (LpL) increased LDL accumulation 5-fold (0.1 +/- 0.03 mV/min [control] versus 0.5 +/- 0.05 mV/min [LpL]) by increasing LDL retention in the artery wall. This effect was blocked by heparin and monoclonal antibodies directed against the amino-terminal region of apolipoprotein B (apo B). This suggests that specific regions of apo B are involved in LDL accumulation within arteries. Also, the effect of hydrolysis of triglyceride-rich lipoproteins on endothelial barrier function was studied. We compared endothelial layer permeability using a water-soluble reference molecule, fluorescently labeled dextran. When LpL was added to hypertriglyceridemic plasma, dextran accumulation within the artery wall increased > 4-fold (0.024 +/- 0.01 mV/min [control] versus 0.098 +/- 0.05 mV/min [LpL]). Under the same conditions, LpL increased LDL accumulation approximately 3-fold (0.016 +/- 0.003 mV/min [control] versus 0.047 +/- 0.013 mV/min [LpL]). Rapid efflux of LDL from the artery wall indicated that increased endothelial layer permeability was the primary mechanism during periods of increased lipolysis. Our data demonstrate two LpL-mediated effects that may increase the amount of LDL in the artery wall. These findings may pertain to the observed relationship between increased postprandial lipemia and atherosclerosis.     

Variation in lipoprotein(a) concentrations among individuals with the same apolipoprotein (a) isoform is determined by the rate of lipoprotein(a) production

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein which is similar in structure to, but metabolically distinct from, LDL. Factors regulating plasma concentrations of Lp(a) are poorly understood. Apo(a), the protein that distinguishes Lp(a) from LDL, is highly polymorphic, and apo(a) size is inversely correlated with plasma Lp(a) level. Even within the same apo(a) isoform class, however, plasma Lp(a) concentrations vary widely. A series of in vivo kinetic studies were performed using purified radiolabeled Lp(a) in individuals with the same apo(a) isoform but different Lp(a) levels. In a group of seven subjects with a single S4-apo(a) isoform and Lp(a) levels ranging from 1 to 13.2 mg/dl, the fractional catabolic rate (FCR) of 131I-labeled S2-Lp(a) (mean 0.328 day-1) was not correlated with the plasma Lp(a) level (r = -0.346, P = 0.45). In two S4-apo(a) subjects with a 10-fold difference in Lp(a) level, the FCR's of 125I-labeled S4-Lp(a) were very similar in both subjects and not substantially different from the FCRs of 131I-S2-Lp(a) in the same subjects. In four subjects with a single S2-apo(a) isoform and Lp(a) levels ranging from 9.4 to 91 mg/dl, Lp(a) concentration was highly correlated with Lp(a) production rate (r = 0.993, P = 0.007), but poorly correlated with Lp(a) FCR (mean 0.304 day-1). Analysis of Lp(a) kinetic parameters in all 11 subjects revealed no significant correlation of Lp(a) level with Lp(a) FCR (r = -0.53, P = 0.09) and a strong correlation with Lp(a) production rate (r = 0.99, P < 0.0001). We conclude that the substantial variation in Lp(a) levels among individuals with the same apo(a) phenotype is caused primarily by differences in Lp(a) production rate.     

Lipoprotein(a): a link between thrombosis and atherosclerosis?

Lipoprotein(a) (Lp(a)) represents a class of plasma lipoproteins similar to low-density lipoprotein (LDL), but containing an unique apolipoprotein(a) with striking homology to plasminogen. Plasma Lp(a) is inherited as a quantitative genetic trait, with a continuous distribution in Caucasian populations (< 10-2000 mg/l), where high levels are associated with an increased risk of atherosclerotic disease. The physiological role of Lp(a) is unknown, and the metabolism is obscure. Plasma Lp(a) is apparently resistent to diets and drug therapy, and LDL-apheresis is currently the most effective way of reducing plasma Lp(a). However, clinical benefits of lowering plasma Lp(a) have not been demonstrated, and specific therapeutic goals cannot be recommended at present. The structural similarity between apo(a) and plasminogen has generated several experimental observations indicating a prothombogenic and proatherogenic role of Lp(a), but the exact pathophysiological mechanisms have not been determined.     

Amyloid beta-peptide spin trapping. II: Evidence for decomposition of the PBN spin adduct

We investigated the effect of lipoprotein(a) (Lp(a)) on proliferation of human arterial smooth muscle cells (SMCs) and its mechanisms of action. Low density lipoprotein (LDL), Lp(a) and apolipoprotein(a) (apo(a)) significantly stimulated the proliferation of SMCs. Lp(a) and apo(a) reduced the amount of active transforming growth factor-beta (TGF-beta) with the mink lung epithelial cell bioassay, however LDL had no effect. Lp(a), but not apo(a), significantly stimulated the proliferation of SMCs even in the presence of a neutralizing antibody for TGF-beta. Our results suggest that Lp(a) stimulates the proliferation of SMCs via apo(a)-induced inhibition of TGF-beta activation and stimulation of SMCs by the LDL-particle of Lp(a).