Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease of patients with diabetes and renal failure, in part by preventing apolipoprotein B (apoB)-mediated cellular uptake of low density lipoproteins (LDL) by LDL receptors (LDLr). It has been proposed that AGE modification at one site in apoB, almost 1,800 residues from the putative apoB LDLr-binding domain, may be sufficient to induce an apoB conformational change that prevents binding to the LDLr. To further explore this hypothesis, we used 29 anti-human apoB mAbs to identify other potential sites on apoB that may be modified by in vitro advanced glycation of LDL. Glycation of LDL caused a time-dependent decrease in its ability to bind to the LDLr and in the immunoreactivity of six distinct apoB epitopes, including two that flank the apoB LDLr-binding domain. ApoB appears to be modified at multiple sites by these criteria, as the loss of glycation-sensitive epitopes was detected on both native glycated LDL and denatured, delipidated glycated apoB. Moreover, residues directly within the putative apoB LDLr-binding site are not apparently modified in glycated LDL. We propose that the inability of LDL modified by AGEs to bind to the LDLr is caused by modification of residues adjacent to the putative LDLr-binding site that were undetected by previous immunochemical studies. AGE modification either eliminates the direct participation of the residues in LDLr binding or indirectly alters the conformation of the apoB LDLr-binding site.

Apolipoprotein E competitively inhibits receptor-dependent low density lipoprotein uptake by the liver but has no effect on cholesterol absorption or synthesis in the mouse.

This study examines the question of whether apolipoprotein E (apoE) alters steady-state concentrations of plasma cholesterol carried in low density lipoproteins (LDL-C) by acting as a competitive inhibitor of hepatic LDL uptake or by altering the rate of net cholesterol delivery from the intestinal lumen to the liver. To differentiate between these two possibilities, rates of cholesterol absorption and synthesis and the kinetics of hepatic LDL-C transport were measured in vivo in mice with either normal (apoE+/+) or zero (apoE-/-) levels of circulating apoE. Rates of cholesterol absorption were essentially identical in both genotypes and equaled approximately 44% of the daily dietary load of cholesterol. This finding was consistent with the further observation that the rates of cholesterol synthesis in the liver (approximately 2,000 nmol/h) and extrahepatic tissues (approximately 3,000 nmol/h) were also essentially identical in the two groups of mice. However, the apparent Michaelis constant for receptor-dependent hepatic LDL-C uptake was markedly lower in the apoE-/- mice (44 +/- 4 mg/dl) than in the apoE+/+ animals (329 +/- 77 mg/dl) even though the maximal transport velocity for this uptake process was essentially the same (approximately 400 micrograms/h per g) in the two groups of mice. These studies, therefore, demonstrate that apoE-containing lipoproteins can act as potent competitive inhibitors of hepatic LDL-C transport and so can significantly increase steady-state plasma LDL-C levels. This apolipoprotein plays no role, however, in the regulation of cholesterol absorption, sterol biosynthesis, or hepatic LDL receptor number, at least in the mouse.

The roles of coenzyme Q10 and vitamin E on the peroxidation of human low density lipoprotein subfractions.

The aim of our study was to investigate the relationships between the levels of coenzyme Q10 (CoQ10) and vitamin E and the levels of hydroperoxide in three subfractions of low density lipoproteins (LDL) that were isolated from healthy donors. LDL3, the densest of the three subfractions, has shown statistically significant lower levels of CoQ10 and vitamin E, which were associated with higher hydroperoxide levels when compared with the lighter counterparts. After CoQ10 supplementation, all three LDL subfractions had significantly increased CoQ10 levels. In particular, LDL3 showed the highest CoQ10 increase when compared with LDL1 and LDL2 and was associated with a significant decrease in hydroperoxide level. These results support the hypothesis that the CoQ10 endowment in subfractions of LDL affects their oxidizability, and they have important implications for the treatment of disease.

Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor.

The very low density lipoprotein (VLDL) receptor is a recently cloned member of the low density lipoprotein (LDL) receptor family that mediates the binding and uptake of VLDL when overexpressed in animal cells. Its sequence is 94% identical in humans and rabbits and 84% identical in humans and chickens, implying a conserved function. Its high level expression in muscle and adipose tissue suggests a role in VLDL triacylglycerol delivery. Mutations in the chicken homologue cause female sterility, owing to impaired VLDL and vitellogenin uptake during egg yolk formation. We used homologous recombination in mouse embryonic stem cells to produce homozygous knockout mice that lack immunodetectable VLDL receptors. Homozygous mice of both sexes were viable and normally fertile. Plasma levels of cholesterol, triacylglycerol, and lipoproteins were normal when the mice were fed normal, high-carbohydrate, or high-fat diets. The sole abnormality detected was a modest decrease in body weight, body mass index, and adipose tissue mass as determined by the weights of epididymal fat pads. We conclude that the VLDL receptor is not required for VLDL clearance from plasma or for ovulation in mice.

Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: Direct evidence that lipoprotein lipase bridging occurs in vivo

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7×; LPL1, 1.8×), core cholesteryl ether (LPL2, 2.3×; LPL1, 2.7×), and apolipoprotein (LPL1, 1.8×; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.

Suppression of diet-induced atherosclerosis in low density lipoprotein receptor knockout mice overexpressing lipoprotein lipase.

Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of triglyceride-rich lipoproteins. Conflicting results have been reported concerning its role in atherogenesis. To determine the effects of the overexpressed LPL on diet-induced atherosclerosis, we have generated low density lipoprotein receptor (LDLR) knockout mice that overexpressed human LPL transgene (LPL/LDLRKO) and compared their plasma lipoproteins and atherosclerosis with those in nonexpressing LDLR-knockout mice (LDLRKO). On a normal chow diet, LPL/LDLRKO mice showed marked suppression of mean plasma triglyceride levels (32 versus 236 mg/dl) and modest decrease in mean cholesterol levels (300 versus 386 mg/dl) as compared with LDLRKO mice. Larger lipoprotein particles of intermediate density lipoprotein (IDL)/LDL were selectively reduced in LPL/LDLRKO mice. On an atherogenic diet, both mice exhibited severe hypercholesterolemia. But, mean plasma cholesterol levels in LPL/ LDLRKO mice were still suppressed as compared with that in LDLRKO mice (1357 versus 2187 mg/dl). Marked reduction in a larger subfraction of IDL/LDL, which conceivably corresponds to remnant lipoproteins, was observed in the LPL/LDLRKO mice. LDLRKO mice developed severe fatty streak lesions in the aortic sinus after feeding with the atherogenic diet for 8 weeks. In contrast, mean lesion area in the LPL/LDLRKO mice was 18-fold smaller than that in LDLRKO mice. We suggest that the altered lipoprotein profile, in particular the reduced level of remnant lipoproteins, is mainly responsible for the protection by LPL against atherosclerosis.

Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line.

The epsilon 4 allele of apolipoprotein E (apoE) is a major risk factor for Alzheimer disease, suggesting that apoE may directly influence neurons in the aging brain. Recent data suggest that apoE-containing lipoproteins can influence neurite outgrowth in an isoform-specific fashion. The neuronal mediators of apoE effects have not been clarified. We show here that in a central nervous system-derived neuronal cell line, apoE3 but not apoE4 increases neurite extension. The effect of apoE3 was blocked at low nanomolar concentrations by purified 39-kDa protein that regulates ligand binding to the low density lipoprotein receptor-related protein (LRP). Anti-LRP antibody also completely abolished the neurite-promoting effect of apoE3. Understanding isoform-specific cell biological processes mediated by apoE-LRP interactions in central nervous system neurons may provide insight into Alzheimer disease pathogenesis.

Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor plays a central role in mammalian cholesterol metabolism, clearing lipoproteins which bear apolipoproteins E and B-100 from plasma. Mutations in this molecule are associated with familial hypercholesterolemia, a condition which leads to an elevated plasma cholesterol concentration and accelerated atherosclerosis. The N-terminal segment of the LDL receptor contains a heptad of cysteine-rich repeats that bind the lipoproteins. Similar repeats are present in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement. The first repeat of the human LDL receptor has been expressed in Escherichia coli as a glutathione S-transferase fusion protein, and the cleaved and purified receptor module has been shown to fold to a single, fully oxidized form that is recognized by the monoclonal antibody IgG-C7 in the presence of calcium ions. The three-dimensional structure of this module has been determined by two-dimensional NMR spectroscopy and shown to consist of a beta-hairpin structure, followed by a series of beta turns. Many of the side chains of the acidic residues, including the highly conserved Ser-Asp-Glu triad, are clustered on one face of the module. To our knowledge, this structure has not previously been described in any other protein and may represent a structural paradigm both for the other modules in the LDL receptor and for the homologous domains of several other proteins. Calcium ions had only minor effects on the CD spectrum and no effect on the 1H NMR spectrum of the repeat, suggesting that they induce no significant conformational change.

Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivo.

The 39-kDa receptor-associated protein (RAP) associates with the multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) and thereby prevents the binding of all known ligands, including alpha 2-macroglobulin and chylomicron remnants. RAP is predominantly localized in the endoplasmic reticulum, raising the possibility that it functions as a chaperone or escort protein in the biosynthesis or intracellular transport of LRP. Here we have used gene targeting to show that RAP promotes the expression of functional LRP in vivo. The amount of mature, processed LRP is reduced in liver and brain of RAP-deficient mice. As a result, hepatic clearance of alpha 2-macroglobulin is impaired and remnant lipoproteins accumulate in the plasma of RAP-deficient mice that also lack functional LDL receptors. These results are consistent with the hypothesis that RAP stabilizes LRP within the secretory pathway. They also suggest a further mechanism by which the activity of an endocytic receptor may be modulated in vivo.

Initial hepatic removal of chylomicron remnants is unaffected but endocytosis is delayed in mice lacking the low density lipoprotein receptor.

Two endocytic receptors, the low density lipoprotein (LDL) receptor (LDLR) and the LDLR-related protein (LRP), are thought to act in concert in the hepatic uptake of partially metabolized dietary lipoproteins, the chylomicron remnants. We have evaluated the role of these two receptors in the hepatic metabolism of chylomicron remnants in normal mice and in LDLR-deficient [LDLR (-/-)] mice. The rate of chylomicron remnant removal by the liver was normal up to 30 min after intravenous injection of chylomicrons into LDLR (-/-) mice and was unaffected by receptor-associated protein (RAP), a potent inhibitor of ligand binding to LRP. In contrast, endocytosis of the remnants by the hepatocytes, measured by their accumulation in the endosomal fraction and by the rate of hydrolysis of component cholesteryl esters, was dramatically reduced in the absence of the LDLR. Coadministration of RAP prevented the continuing hepatic removal of chylomicron remnants in LDL (-/-) mice after 30 min, consistent with blockade of the slow endocytosis by a RAP-sensitive process. Taken together with previous studies, our results are consistent with a model in which the initial hepatic removal of chylomicron remnants is primarily mediated by mechanisms that do not include LDLR or LRP, possibly involving glycosaminoglycan-bound hepatic lipase and apolipoprotein E. After the remnants bind to these alternative sites on the hepatocyte surface, endocytosis is predominantly mediated by the LDLR and also by a slower and less efficient backup process that is RAP sensitive and therefore most likely involves LRP.

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 ≈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.

Expression of the peroxisome proliferator-activated receptor γ (PPARγ) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein

The peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARγ is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARγ is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARγ expression in primary macrophages and monocytic cell lines. PPARγ mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARγ expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARγ expression in macrophages. TPA induced the expression of PPARγ in RAW 264.7 macrophages by increasing transcription from the PPARγ1 and PPARγ3 promoters. In concert, these observations provide insights into the regulation of PPARγ expression in activated macrophages and raise the possibility that PPARγ ligands may influence the progression of atherosclerosis.

Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor

Endocytosis of the Flaviviridae viruses, hepatitis C virus, GB virus C/hepatitis G virus, and bovine viral diarrheal virus (BVDV) was shown to be mediated by low density lipoprotein (LDL) receptors on cultured cells by several lines of evidence: by the demonstration that endocytosis of these virus correlated with LDL receptor activity, by complete inhibition of detectable endocytosis by anti-LDL receptor antibody, by inhibition with anti-apolipoprotein E and -apolipoprotein B antibodies, by chemical methods abrogating lipoprotein/LDL receptor interactions, and by inhibition with the endocytosis inhibitor phenylarsine oxide. Confirmatory evidence was provided by the lack of detectable LDL receptor on cells known to be resistant to BVDV infection. Endocytosis via the LDL receptor was shown to be mediated by complexing of the virus to very low density lipoprotein or LDL but not high density lipoprotein. Studies using LDL receptor-deficient cells or a cytolytic BVDV system indicated that the LDL receptor may be the main but not exclusive means of cell entry of these viruses. Studies on other types of viruses indicated that this mechanism may not be exclusive to Flaviviridae but may be used by viruses that associate with lipoprotein in the blood. These findings provide evidence that the family of LDL receptors may serve as viral receptors.

Two sterol regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lipoprotein free cholesterol

Caveolae form the terminus for a major pathway of intracellular free cholesterol (FC) transport. Caveolin mRNA levels in confluent human skin fibroblasts were up-regulated following increased uptake of low density lipoprotein (LDL) FC. The increase induced by FC was not associated with detectable change in mRNA stability, indicating that caveolin mRNA levels were mediated at the level of gene transcription. A total of 924 bp of 5′ flanking region of the caveolin gene were cloned and sequenced. The promoter sequence included three G+C-rich potential sterol regulatory elements (SREs), a CAAT sequence and a Sp1 consensus sequence. Deletional mutagenesis of individual SRE-like sequences indicated that of these two (at −646 and −395 bp) were essential for the increased transcription rates mediated by LDL-FC, whereas the third was inconsequential. Gel shift analysis of protein binding from nuclear extracts to these caveolin promoter DNA sequences, together with DNase I footprinting, confirmed nucleoprotein binding to the SRE-like elements as part of the transcriptional response to LDL-FC. A supershift obtained with antibody to SRE-binding protein 1 (SPEBP-1) indicated that this protein binds at −395 bp. There was no reaction at −395 bp with anti-Sp1 antibody nor with either antibody at −646 bp. The cysteine protease inhibitor N-acetyl-leu-leu-norleucinal (ALLN), which inhibits SREBP catabolism, superinhibited caveolin mRNA levels regardless of LDL-FC. This finding suggests that SREBP inhibits caveolin gene transcription in contrast to its stimulating effect on other promoters. The findings of this study are consistent with the postulated role for caveolin as a regulator of cellular FC homeostasis in quiescent peripheral cells, and the coordinate regulation by SREBP of FC influx and efflux.