Apolipoprotein E binding to low density lipoprotein receptor-related protein-1 inhibits cell migration via activation of cAMP-dependent protein kinase A

Smooth muscle cell migration and proliferation contribute to neointimal hyperplasia and vascular stenosis after endothelial denudation. Previous studies revealed that apolipoprotein E (apoE) is an effective inhibitor of platelet-derived growth factor-directed smooth muscle cell migration and proliferation and that the anti-migratory function is mediated via apoE binding to low density lipoprotein receptor-related protein-1 (LRP-1). This study was undertaken to identify the intracellular pathway by which apoE binding to LRP-1 results in inhibition of smooth muscle cell migration. The results showed that apoE increased intracellular cAMP levels 3-fold after 5 min, and the increase was sustained for more than 1 h. As a consequence, apoE also increased protein kinase A (PKA) activity in smooth muscle cells. Importantly, suppression of PKA activity with a cell-permeable peptide inhibitor of PKA abolished the inhibitory effect of apoE on smooth muscle cell migration. These results indicated that apoE inhibition of smooth muscle cell migration is mediated via the activation of cAMP-dependent PKA. Additional experiments revealed that apoE also inhibited fibroblasts migration toward platelet-derived growth factor by a similar mechanism of cAMP-dependent PKA activation. It is noteworthy that apoE failed to increase cAMP levels or inhibit migration of LRP-1-negative mouse embryonic fibroblasts and LRP-1-deficient smooth muscle cells. Taken together, these findings established the mechanism by which apoE inhibits cell migration, i.e. via cAMP-dependent protein kinase A activation as a consequence of its binding to LRP-1.     

Apolipoprotein E-mediated binding of hypertriglyceridemic very low density lipoproteins to isolated low density lipoprotein receptors detected by ligand blotting

HTG-VLDL1, like LDL, bind with high affinity to electrophoretically transferred, isolated LDL receptors partially purified from bovine adrenal glands. Ligand blotting techniques show that binding is calcium dependent; little or no binding of LDL or HTG-VLDL1 is observed in the presence of 10 mM EDTA. HTG-VLDL1 does not bind in the presence of 7 mM suramin, an inhibitor of LDL binding to the LDL receptor. Pretreatment of LDL with either thrombin or trypsin does not affect apoB-mediated LDL binding to the LDL receptor. ApoE-mediated binding of HTG-VLDL1 to the blotted LDL receptor is abolished or greatly decreased by thrombin treatment of HTG-VLDL1; trypsin treatment of HTG-VLDL1 abolishes binding. Reincorporation of apoE into trypsinized HTG-VLDL1 restores binding. These studies demonstrate unequivocally that HTG-VLDL1 bind to the LDL receptor, that the binding of HTG-VLDL1 to the isolated LDL receptor is mediated through the thrombin-accessible apoE, and that HTG-VLDL1 which bind via potentially dissociable apoE rather than non-transferable apoB can be used for ligand blotting.     

Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) from rat liver membranes binds apoprotein E (apoE)-enriched rabbit beta-migrating very low density lipoproteins (beta-VLDL) in a ligand blotting assay on nitrocellulose membranes. Binding was markedly activated when the beta-VLDL was preincubated with recombinant human apoE-3, native human apoE-3 or E-4, or native rabbit apoE. Human apoE-2, which binds poorly (1-2% of apo E-3 binding) to low density lipoprotein receptors, was approximately 40% as effective as apoE-3 or apoE-4 in binding to LRP. Stimulation of apoE-dependent binding to LRP was blocked by the inclusion of a mixture of human apoC proteins, but not apoA-I or A-II, in the preincubation reaction. High concentrations of apoE did not overcome the apoC inhibition. The effects of apoE and apoC on the ligand blotting assay were paralleled by similar effects in the ability of beta-VLDL to stimulate cholesteryl ester synthesis in mutant human fibroblasts that lack low density lipoprotein receptors. These properties of LRP are consistent with the known effects of apoE and apoC on uptake of chylomicron and very low density lipoprotein remnants in the liver and raise the possibility that LRP functions as a receptor for apoE-enriched forms of these lipoproteins in intact animals.     

Structural peculiarities of the binding of very low density lipoproteins and low density lipoproteins to the LDL receptor in hypertriglyceridemia: role of apolipoprotein E

Very low (VLDL) and low density lipoproteins (LDL) were isolated from plasma of patients with the E3/3 phenotype which were divided into three groups based on their plasma triglyceride content: low (TG<200 mg/dl, TG(l)), intermediate (200<300 mg/dl, TG(i)300 mg/dl, TG(h)). The protein density (PD) on the VLDL and LDL surface was calculated from lipoprotein composition and protein location was studied by tryptophan fluorescence quenching by I(-) anions at 25 degrees C and 40 degrees C. A comparison of the TG(h) with the TG(l) group revealed a significant (<0.05) increase of the PD parameter as much as 21% for VLDL, but not for LDL where this parameter did not change for any group; generally, PD(LDL) values were 3.2-3.8-fold lower than PD(VLDL). In accordance with this difference, the tryptophan accessibility f in VLDL vs. LDL was lower at both temperatures. There were temperature-induced changes of the f parameter in opposite directions for these lipoproteins. The difference in f value gradually decreased for VLDL in the direction TG(l)TG(i)TG(h) while for LDL there was a U-shaped dependence for these groups. The Stern-Volmer quenching constant K(S-V) which is sensitive to both temperature and viscosity, did not change for VLDL, but K(S-V)(LDL) was 2-3-fold higher for the TG(i) group compared to the other two. The efficiencies of VLDL and LDL binding to the LDL receptor (LDLr) in vitro were compared by solid-phase assay free of steric hindrance observed in cell binding. The maximal number of binding sites did not change for either type of particles and between groups. The association constant K(a) and apolipoprotein (apo) E/apoB mole ratio values all increased significantly for VLDL, but not for LDL, in comparison of the TG(i+h) with the TG(l) group. Based on VLDL and LDL concentrations in serum and on the affinity constant values obtained in an in vitro assay, VLDL concentrations corresponding to 50% inhibition of LDL binding (IC(50)) were calculated in an assumption of the competition of both ligands for LDLr in vivo; the mean values of IC(50) decreased 2-fold when plasma TG exceeded 200 mg/dl. The functional dependences of K(a)(VLDL), IC(50) and apoE content in VLDL (both fractional and absolute) and in serum on TG content in the whole concentration range studied were fitted to a saturation model. For all five parameters, the mean half-maximum values TG(1/2) were in the range 52-103 mg/dl. The efficiency of protein-protein interactions is suggested to differ in normolipidemic vs. HTG-VLDL and apoE content and/or protein density on VLDL surface may be the primary determinant(s) of the increased binding of HTG-VLDL to the LDL receptor. ApoCs may compete with apoE for the binding to the VLDL lipid surface as plasma triglyceride content increases. The possible competition of VLDL with LDL for the catabolism site(s) in vivo, when plasma TG increases, could explain the atherogenic action of TG-rich lipoproteins. Moreover, the 'dual action' hypothesis on anti-atherogenic action of apoE-containing high density lipoproteins (HDL) in vivo is suggested: besides the well-known effect of HDL as cholesteryl ester catabolic outway, the formation of a transient complex of apoE-containing discs appearing at the site of VLDL TG hydrolysis by lipoprotein lipase with VLDL particles proposed in our preceding paper promotes the efficient uptake of TG-rich particles; in hypertriglyceridemia due to the diminished HDL content this uptake seems to be impaired which results in the increased accumulation of the remnants of TG-rich particles. This explains the observed increase in cholesterol and triglyceride content in VLDL and LDL, respectively, due to the CETP-mediated exchange of cholesteryl ester and triglyceride molecules between these particles.     

A change in apolipoprotein B expression is required for the binding of apolipoprotein E to very low density lipoprotein

Factors affecting the association of apolipoprotein E (apoE) with human plasma very low density lipoprotein (VLDL) were investigated in experiments in which the lipid content of the lipoprotein was modified either by lipid transfer in the absence of lipolysis or through the action of lipoprotein lipase. In both cases, lipoprotein particles initially containing no apoE (VLDL-E), isolated by heparin affinity chromatography, were modified until they had the same lipid composition as native apoE-containing VLDL (VLDL+E) from the same plasma. Transfer-modified lipoproteins, unlike native VLDL+E, did not bind apoE or interact with heparin. In contrast, VLDL-E, whose lipid composition was modified to the same extent by lipase, bound apoE and bound to heparin under the same conditions as native VLDL+E. A structural protein (apolipoprotein B) epitope characteristic of VLDL+E was expressed during lipolysis prior to ApoE or heparin binding. The data suggest that the reaction of apoE with VLDL-E is a two-step reaction. The appearance of apoB is modified during lipolysis, with expression of a major heparin-binding site. The modified VLDL then becomes competent to bind apoE. The lipid composition of VLDL appears not to be a major factor in the ability of VLDL to bind apoE or to bind to heparin.     

Apolipoprotein E and low density lipoprotein receptor-related protein facilitate intraneuronal Abeta42 accumulation in amyloid model mice

The low density lipoprotein receptor-related protein (LRP) is highly expressed in the brain and has been shown to alter the metabolism of amyloid precursor protein and amyloid-beta peptide (Abeta) in vitro. Previously we developed mice that overexpress a functional LRP minireceptor (mLRP2) in their brains and crossed them to the PDAPP mouse model of Alzheimer disease. Overexpression of mLRP2 in 22-month-old PDAPP mice with amyloid plaques increased a pool of carbonate-soluble Abeta in the brain and worsened memory-related behavior. In the current study, we examined the effects of mLRP2 overexpression on 3-month-old PDAPP mice that had not yet developed amyloid plaques. We found significantly higher levels of membrane-associated Abeta42 in the hippocampus of mice that overexpressed mLRP2. Using immunohistochemical methods, we observed significant intraneuronal Abeta42 in the hippocampus and frontal cortex of PDAPP mice, which frequently co-localized with the lysosomal marker LAMP-1. Interestingly, PDAPP mice lacking apolipoprotein E (apoE) had much less intraneuronal Abeta42. We also found that PC12 cells overexpressing mLRP2 cleared Abeta42 and Abeta40 more rapidly from media than PC12 cells transfected with the vector only. Preincubation of apoE3 or apoE4 with Abeta42 increased the rate of Abeta clearance, and this effect was partially blocked by receptor-associated protein. Our results support the hypothesis that LRP binds and endocytoses Abeta42 both directly and via apoE but that endocytosed Abeta42 is not completely degraded and accumulates in intraneuronal lysosomes.     

39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.     

Interactions of low density lipoprotein2 and other apolipoprotein B-containing lipoproteins with lipoprotein(a)

Studies were undertaken to investigate potential interactions among plasma lipoproteins. Techniques used were low density lipoprotein2 (LDL2)-ligand blotting of plasma lipoproteins separated by nondenaturing 2.5-15% gradient gel electrophoresis, ligand binding of plasma lipoproteins by affinity chromatography with either LDL2 or lipoprotein(a) (Lp(a)) as ligands, and agarose lipoprotein electrophoresis. Ligand blotting showed that LDL2 can bind to Lp(a). When apolipoprotein(a) was removed from Lp(a) by reduction and ultracentrifugation, no interaction between LDL2 and reduced Lp(a) was detected by ligand blotting. Ligand binding showed that LDL2-Sepharose 4B columns bound plasma lipoproteins containing apolipoproteins(a), B, and other apolipoproteins. The Lp(a)-Sepharose column bound lipoproteins containing apolipoprotein B and other apolipoproteins. Furthermore, the Lp(a) ligand column bound more lipoprotein lipid than the LDL2 ligand column, with the Lp(a) ligand column having a greater affinity for triglyceride-rich lipoproteins. Lipoprotein electrophoresis of a mixture of LDL2 and Lp(a) demonstrated a single band with a mobility intermediate between that of LDL2 and Lp(a). Chemical modification of the lysine residues of apolipoprotein B (apoB) by either acetylation or acetoacetylation prevented or diminished the interaction of LDL2 with Lp(a), as shown by both agarose electrophoresis and ligand blotting using modified LDL2. Moreover, removal of the acetoacetyl group from the lysine residues of apoB by hydroxylamine reestablished the interaction of LDL2 with Lp(a). On the other hand, blocking of--SH groups of apoB by iodoacetamide failed to show any effect on the interaction between LDL2 and Lp(a). Based on these observations, it was concluded that Lp(a) interacts with LDL2 and other apoB-containing lipoproteins which are enriched in triglyceride; this interaction is due to the presence of apolipoprotein(a) and involves lysine residues of apoB interacting with the plasminogen-like domains (kringle 4) of apolipoprotein(a). Such results suggest that Lp(a) may be involved in triglyceride-rich lipoprotein metabolism, could form transient associations with apoB-containing lipoproteins in the vascular compartment, and alter the intake by the high affinity apoB, E receptor pathway.     

Receptor-associated protein binding blocks ubiquitinylation of the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) consists of two subunits, M(r) approximately 515,000 and 85,000. LRP is a receptor for activated alpha2-macrogobulin (alpha2M*), Pseudomonas exotoxin A, and many other proteins. We now report that ubiquitinylation of the LRP heavy chain occurred when either Pseudomonas exotoxin A or alpha2M* bound to LRP on macrophages. Ubiquitinylation was dose-dependent and maximal about 30 min after ligation of the receptor. Addition of the proteosome inhibitor MG-132 sustained the level of ubiquitin-LRP for longer time intervals in macrophages treated with either alpha2M* or Pseudomonas exotoxin A. By contrast, when receptor associated protein (RAP) bound to LRP, ubiquitinylation did not occur. While RAP is not found in the extracellular environment it binds to LRP and is believed to function as an intracellular chaperone. The presence of RAP within the cell may, therefore, contribute to the recycling of intact LRP which has ligated and internalized its ligands.     

Apolipoprotein E Bethesda. Isolation and partial characterization of a variant of human apolipoprotein E isolated from very low density lipoproteins

A variant of apolipoprotein E, denoted E Bethesda, has been identified in the plasma of a 72-year-old woman with type III hyperlipoproteinemia. An offspring of the proband also has this variant and type III hyperlipoproteinemia. Apolipoprotein E Bethesda was isolated by preparative isoelectrofocusing followed by preparative SDS-polyacrylamide gel electrophoresis from the very low density lipoproteins of the proband's son. The purity and the identity of the preparation were analyzed by analytical SDS-polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis and by immunochemical analysis. Apolipoprotein E Bethesda migrates in the E 1 position and its electrophoretic mobility is not affected by neuraminidase treatment. The protein is shifted to the E3 position after cysteamine treatment. The amino acid composition revealed the presence of two cysteine residues. These data support the concept that the apolipoprotein E Bethesda allele is derived from a mutation of the E2 or E2* allele.     

A potential neuroprotective role of apolipoprotein E-containing lipoproteins through low density lipoprotein receptor-related protein 1 in normal tension glaucoma

Glaucoma is an optic neuropathy and the second major cause of blindness worldwide next to cataracts. The protection from retinal ganglion cell (RGC) loss, one of the main characteristics of glaucoma, would be a straightforward treatment for this disorder. However, the clinical application of neuroprotection has not, so far, been successful. Here, we report that apolipoprotein E-containing lipoproteins (E-LPs) protect primary cultured RGCs from Ca(2+)-dependent, and mitochondrion-mediated, apoptosis induced by glutamate. Binding of E-LPs to the low density lipoprotein receptor-related protein 1 recruited the N-methyl-d-aspartate receptor, blocked intracellular Ca(2+) elevation, and inactivated glycogen synthase kinase 3β, thereby inhibiting apoptosis. When compared with contralateral eyes treated with phosphate-buffered saline, intravitreal administration of E-LPs protected against RGC loss in glutamate aspartate transporter-deficient mice, a model of normal tension glaucoma that causes glaucomatous optic neuropathy without elevation of intraocular pressure. Although the presence of α2-macroglobulin, another ligand of the low density lipoprotein receptor-related protein 1, interfered with the neuroprotective effect of E-LPs against glutamate-induced neurotoxicity, the addition of E-LPs overcame the inhibitory effect of α2-macroglobulin. These findings may provide a potential therapeutic strategy for normal tension glaucoma by an LRP1-mediated pathway.     

Thf complete amino acid sequence of C-I (apoLp-Ser), an apolipoprotein from human very low density lipoproteins.

C-I was prepared from very low density lipoproteins of patients with familial type V hyperliporproteinemia. Peptides from tryptic digests of unmodified and succinylated C-I, chymotryptic peptides, and the products of cayanogen bromide cleavage were isolated and characterized. Sequence analysis of tryptic peptides was performed by the dansyl (5-dimethylaminonaphthalene-1-sulfonyl) technique and hydrolytic regeneration of the amino acid residues from the phenylthiocarbamyl derivatives. Alignment of the tryptic fragments within the cyanogen bromide and succinyl-tryptic peptides was confirmed by the overlap chymotryptic peptides. The complete amino acid sequence of C-I, 57 residues in length, does not reveal any obvious basis for its lipophilic properties.     

Low density lipoprotein receptor-related protein 1 dependent endosomal trapping and recycling of apolipoprotein E

Background

Lipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling.

Principal Findings

Immunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion.

Conclusion

We conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles.     

Heparin binding to lipoprotein lipase and low density lipoproteins

Heparin was fractionated on an affinity column of bovine milk lipoprotein lipase (LpL) immobilized to Affi-Gel-15. The bound heparin, designated high-reactive heparin (HRH), enhanced LpL activity, presumably by stabilizing the enzyme against denaturation. The unbound heparin fraction had no observable effect on the initial rate of enzyme activity. However, at longer times of incubation there was inhibition of LpL activity. LpL-specific HRH also showed a high, Ca²⁺-dependent precipitating activity towards human plasma low density lipoproteins (LDL). Since LpL and LDL both bind to heparin-like molecules at the surface of the arterial wall, we suggest that their similar heparin-binding specificity may have physiological consequences as it relates to the development of atherosclerosis.

Heparin binding Lipoprotein lipase LDL Apolipoprotein Lipolysis     

Apolipoprotein E mediates binding of normal very low density lipoprotein to heparin but is not required for high affinity receptor binding

The relationship between the cholesteryl ester content of normal human very low density lipoprotein (VLDL) and its ability to bind to apolipoprotein E (apoE), heparin, and the low density lipoprotein (LDL) receptor have been compared. Plasma VLDL were separated by heparin affinity chromatography into two fractions: one with apoE and one without. Both fractions had the same cholesteryl ester content relative to apolipoprotein B (apoB). LDL, on the other hand, had a greater cholesteryl ester content. VLDL were modified by lipolysis to express the ability to bind apoE (Ishikawa, Y., Fielding, C. J., and Fielding, P. E. (1988) J. Biol. Chem. 263, 2744-2749). Lipolyzed VLDL with or without apoE were compared for their ability to bind to heparin or the up-regulated fibroblast LDL receptor. Lipolyzed VLDL bound with the same affinity to the receptor whether or not the particles contained apoE. ApoB, not apoE, appears then to be the important ligand for normal VLDL. On the other hand, modified VLDL without apoE, even though binding to the LDL receptor, did not bind to heparin. These data suggest that apoE mediates heparin binding in normal VLDL, that apoB mediates receptor binding, and that the cholesteryl ester content of VLDL is not a factor in the induction of the ability to bind apoE.     

Stimulation of the in vivo production of very low density lipoproteins by apolipoprotein E is independent of the presence of the low density lipoprotein receptor

Apolipoprotein (apo) E stimulates the secretion of very low density lipoproteins (VLDLs) by an as yet unknown mechanism. Recently, a working mechanism for apoE was proposed (Twisk, J., Gillian-Daniel, D. L., Tebon, A., Wang, L., Barrett, P. H., and Attie, A. D. (2000) J. Clin. Invest. 105, 521-532) in which apoE prevents the inhibitory action of the low density lipoprotein receptor (LDLr) by binding to it. We have first tested whether this newly described effect of the LDLr on VLDL secretion, obtained in vitro, is also observed in vivo. In LDLr knockout mice (LDLr-/-), the production of VLDL triglycerides and apoB was 30% higher than that in controls. Also the ratio of apoB100:apoB48 secretion was increased in the LDLr-/- mice. The composition of nascent VLDL was similar in both strains. To test whether the action of apoE depends on the presence of the LDLr, VLDL production was measured in LDLr-/- and apoE-/- LDLr-/- mice. Deletion of apoE on a LDLr-/- background still caused a 50% decrease of VLDL triglycerides and apoB production. The composition of nascent VLDL was again similar for both strains. We conclude that the effect of apoE on hepatic VLDL production is independent of the presence of the LDLr.     

The carbohydrate content of apolipoprotein E from human very low density lipoproteins.

The carbohydrate content of the E protein of human very low density lipoprotein (VLDL) was evaluated both by colorimetric methods and by gas liquid chromatography of the trifluoroacetylated 0-methyl glycosides. The major unmodified hexose was noted to be galactose with a mole ratio with respect to protein which ranged from 0.81 to 1.54. N-acetyl glucosamine (molar ratios from 0.52 to 1.76) and N-acetyl galactosamine (molar ratios from 0.73 to 1.59) and the respective unacetylated amino sugars were noted for all of the apoproteins evaluated. Sialic acid (molar ratios from 0.79 to 1.69) was a prominent carbohydrate for each of the E protein preparations. When the apoprotein was exposed to neuraminidase with a resultant loss of two-thirds of the sialic acid, the isoelectric focus behavior was found to be unchanged. The E protein isolated from the very low density lipoproteins of Type III patients (dysbetalipoproteinemia) revealed a carbohydrate content similar to the normals or Type IV patients.     

Apolipoprotein E and lipoprotein lipase secreted from human monocyte-derived macrophages modulate very low density lipoprotein uptake

We investigated the roles of lipoprotein lipase and apolipoprotein E (apoE) secreted from human monocyte-derived macrophages in the uptake of very low density lipoproteins (VLDL). ApoCII-deficient VLDL were isolated from a patient with apoCII deficiency. The lipolytic conversion to higher density and the degradation of the apoCII-deficient VLDL by macrophages were very slight, whereas the addition of apoCII enhanced both their conversion and degradation. This suggests that the lipolysis and subsequent conversion of VLDL to lipoproteins of higher density are essential for the VLDL uptake by macrophages. VLDL incubated with macrophages obtained from subjects with E3/3 phenotype (E3/3-macrophages) showed a 17-fold greater affinity in inhibiting the binding of 2 micrograms/ml 125I-low density lipoprotein (LDL) to fibroblasts than native VLDL, whereas the incubation of VLDL with macrophages obtained from a subject with E2/2 phenotype (E2/2-macrophages) did not cause any increase in their affinity. Furthermore, 3 micrograms/ml 125I-VLDL obtained from a subject with E3/3 phenotype were degraded by E3/3-macrophages to a greater extent than by E2/2-macrophages (2-fold), indicating that VLDL uptake is influenced by the phenotype of apoE secreted by macrophages. From these results, we conclude that both lipolysis by lipoprotein lipase and incorporation of apoE secreted from macrophages alter the affinity of VLDL for the LDL receptors on the cells, resulting in facilitation of their receptor-mediated endocytosis.     

The relationship among apolipoprotein(a) polymorphisms, the low-density lipoprotein receptor-related protein, and the very low density lipoprotein receptor genes, and plasma lipoprotein(A) concentration in the Czech population

Increased plasma concentration of lipoprotein(a) [Lp(a)] is an established independent risk factor for coronary artery disease (CAD), which is strongly genetically determined. This study was designed to investigate the relationship between the K-IV and (TTTTA)n apolipoprotein(a) [apo(a), protein; APOA, gene] polymorphisms, as well as the C766T low-density lipoprotein receptor-related protein (LRP) and the (CGG)n very low density lipoprotein receptor (VLDLR) polymorphisms on the one hand, and plasma Lp(a) levels in Czech subjects who underwent coronary angiography on the other hand. The lengths of the alleles of the APOA K-IV and (TTTTA)n polymorphisms were strongly inversely correlated with plasma Lp(a) levels in univariate analysis (r = -0.41, p < 10(-4) and r = -0.20, p < 0.01, respectively). Multivariate analysis revealed significant associations between the APOA polymorphisms studied and plasma Lp(a) levels in subjects expressing only one APOA K-IV allele (p < 10(-6) for K-IV and p < 0.001 for TTTTA). In subjects expressing both APOA K-IV alleles, the multivariate analysis revealed that only the APOA K-IV alleles were inversely correlated with plasma Lp(a) levels (p < 0.001). Associations between both the LRP and VLDLR gene polymorphisms and plasma Lp(a) levels were only of borderline significance (p < 0.06 and p < 0.07, respectively) and were not confirmed in multivariate analysis. In conclusion, both APOA length polymorphisms significantly influenced plasma Lp(a) concentration in the Czech population studied, and this circumstance could explain the association in this population observed earlier between APOA (TTTTA)n polymorphism and CAD (Benes et al. 2000). Only a minor role in the regulation of plasma Lp(a) levels is suggested for the C766T LRP and the (CGG)n VLDLR polymorphisms.