A fast semi-quantitative LC-MS method for measurement of intact apolipoprotein A-I reveals novel proteoforms in serum.

BACKGROUND: Surrogate markers for reverse cholesterol transport (RCT) efficiency such as HDL cholesterol and immune methods for apolipoprotein A-I (ApoA-I) may not fully reflect the actual efficiency of the RCT pathway. Several genetic variants and different posttranslational proteoforms of ApoA-I may unevenly affect the functionality of the HDL particle to efflux cholesterol. A method employing top-down immunoaffinity LC-MS of ApoA-I in order to characterize the most prevalent ApoA-I proteoforms in human plasma is described. METHODS: Diluted plasma was directly injected into a 2D LC-MS system consisting of an affinity column and an analytical column. Enriched ApoA-I fractions were introduced into the MS and intact or fragmented ApoA-I was analyzed. RESULTS: ApoA-I as detected by the described LC-MS method distributes into at least 14 major potential proteoforms exceeding detection limit in human plasma. Substantial amounts of ApoA-I in plasma were found to occur as truncated, oxidized, glycated and glycosylated proteoforms. Levels of glycated ApoA-I distinguished significantly diabetic from non-diabetic samples. In addition novel truncated and glycosylated proteoforms were detected. CONCLUSIONS: ApoA-I proteoforms measured by LC-MS represent a useful approach to augment the clinical picture of ApoA-I and its function in health and disease.

The thyroid receptor ß modulator GC-1 reduces atherosclerosis in ApoE deficient mice.

Thyroid hormone reduces plasma cholesterol and increases expression of low-density lipoprotein receptor (LDL-R) in liver, an effect mediated by thyroid receptor ß (TRß). The selective TRß modulator GC-1 also enhances several steps in reverse cholesterol transport and can decrease serum cholesterol independently of LDL-R. To test whether GC-1 reduces atherosclerosis and to determine which mechanisms are active, we treated ApoE deficient mice with atherogenic diet ± GC-1. GC-1 reduced cholesteryl esters in aorta after 20 weeks. Serum free and esterified cholesterol were reduced after 1 and 10 weeks, but not 20 weeks. Hepatic bile acid synthesis and LDL-R expression was elevated after 1, 10 and 20 weeks, without changes in hepatic de novo cholesterol synthesis. GC-1 increased faecal neutral sterols and reduced serum campesterol after 1 week, indicating reduced intestinal cholesterol absorption. After 20 weeks, GC-1 increased faecal bile acids, but not faecal neutral sterols. Hepatic scavenger receptor B1 (SR-B1) expression was decreased by GC-1. We conclude that GC-1 delays the onset of atherosclerosis in ApoE deficient mice. Since ApoE is needed for hepatic cholesterol reabsorption by LDL-R, this supports the idea that GC-1 reduces serum cholesterol independently of LDL-R by increasing hepatic bile acid synthesis. GC-1 lipid-lowering effects in ApoE deficient mice may also be partly due to reduced intestinal cholesterol absorption. Since reductions in serum cholesterol are reversed at longer times, these GC-1 dependent effects may not be enough for sustained cholesterol reduction in long term treatments.

LXRß activation increases intestinal cholesterol absorption, leading to an atherogenic lipoprotein profile.

OBJECTIVES: Liver X receptors (LXRs) are essential for the regulation of intestinal cholesterol absorption. Because two isoforms exist, LXRα and LXRß, with overlapping but not identical functions, we investigated whether LXRα and LXRß exert different effects on intestinal cholesterol absorption. DESIGN: Wild-type (WT), LXRα(-/-) and LXRß(-/-) mice were fed control diet, 0.2% cholesterol-enriched diet or 0.2% cholesterol-enriched diet plus the LXR agonist GW3965. RESULTS: When fed a control diet, all three genotypes showed similar levels of cholesterol absorption. Of interest, a significant increase in cholesterol absorption was found in the LXRα(-/-) mice, but not in the WT or LXRß(-/-) animals, when fed a diet enriched with 0.2% cholesterol or 0.2% cholesterol + GW3965. Reduced faecal neutral sterol excretion and a hydrophobic bile acid profile were also observed in LXRα(-/-) mice. Greater increases in the apolipoprotein (apo)B-containing lipoproteins in serum were seen in the LXRα(-/-) mice. A 0.2% cholesterol +GW3965 diet suppressed intestinal Npc1l1 protein expression to the same extent for all genotypes, while Abca1 and Abcg5 were elevated to the same degree. CONCLUSIONS: In the intestine, LXRα and LXRß seem to exert similar effects on expression of cholesterol-transporting proteins such as Npc1l1. Selective activation of LXRß may generate effects such as increased cholesterol absorption and elevated serum levels of apoB-containing lipoproteins, which seem to be counteracted by LXRα. Therefore, an intestinal LXRß-specific pathway might exist in terms of cholesterol transportation in addition to the main pathway.

Studies on LXR- and FXR-mediated effects on cholesterol homeostasis in normal and cholic acid-depleted mice.

As previously reported by us, mice with targeted disruption of the CYP8B1 gene (CYP8B1-/-) fail to produce cholic acid (CA), upregulate their bile acid synthesis, reduce the absorption of dietary cholesterol and, after cholesterol feeding, accumulate less liver cholesterol than wild-type (CYP8B1+/+) mice. In the present study, cholesterol-enriched diet (0.5%) or administration of a synthetic liver X receptor (LXR) agonist strongly upregulated CYP7A1 expression in CYP8B1-/- mice, compared to CYP8B1+/+ mice. Cholesterol-fed CYP8B1-/- mice also showed a significant rise in HDL cholesterol and increased levels of liver ABCA1 mRNA. A combined CA (0.25%)/cholesterol (0.5%) diet enhanced absorption of intestinal cholesterol in both groups of mice, increased their liver cholesterol content, and reduced their expression of CYP7A1 mRNA. The ABCG5/G8 liver mRNA was increased in both groups of mice, but cholesterol crystals were only observed in bile from the CYP8B1+/+ mice. The results demonstrate the cholesterol-sparing effects of CA: enhanced absorption and reduced conversion into bile acids. Farnesoid X receptor (FXR)-mediated suppression of CYP7A1 in mice seems to be a predominant mechanism for regulation of bile acid synthesis under normal conditions and, as confirmed, able to override LXR-mediated mechanisms. Interaction between FXR- and LXR-mediated stimuli might also regulate expression of liver ABCG5/G8.

Polymorphism in the coding part of the sterol 12alpha-hydroxylase gene does not explain the marked differences in the ratio of cholic acid and chenodeoxycholic acid in human bile.

OBJECTIVE: In humans, two primary bile acids are synthesized: cholic acid (CA) and chenodeoxycholic acid (CDCA), the first and rate-limiting enzyme being cholesterol 7alpha-hydroxylase (CYP7A1). CA has one more hydroxyl group at position 12alpha. This hydroxylation is carried out by the sterol 12alpha-hydroxylase (CYP8B1). Earlier, we and others have noticed a marked variation in the ratio between CA and CDCA in human bile. The aim of this study was to investigate whether this marked difference could be due to a genetic polymorphism in the gene of the CYP8B1. MATERIAL AND METHODS: Screening for genetic polymorphisms was carried out in a 2.4-kb-long area including the exon and part of the promoter region in subjects who had undergone cholecystectomy earlier, and where bile acid analysis had been performed. Among these subjects those with very high or low CA/CDCA ratios (ranging from 0.9 to 6.8) were investigated. The subjects were all female, normolipidaemic, having normal weight and a normal thyroid function. RESULTS: No polymorphisms were found in the investigated sequence. However, a statistically significant correlation was found between the activity of the CYP7A1 and the ratio between CA and CDCA. The difference in ratio could, at least in part, be explained by the difference in rate of bile acid synthesis. CONCLUSION: The difference in ratio between CA and CDCA cannot be explained by a polymorphism in the coding area of the CYP8B1.

Mild iron overload in patients carrying the HFE S65C gene mutation: a retrospective study in patients with suspected iron overload and healthy controls.

BACKGROUND AND AIMS: The role of the HFE S65C mutation in the development of hepatic iron overload is unknown. The aim of the present study was: (A) to determine the HFE S65C frequency in a Northern European population; and (B) to evaluate whether the presence of the HFE S65C mutation would result in a significant hepatic iron overload. PATIENTS AND METHODS: Biochemical iron parameters and HFE mutation analysis (for the C282Y, H63D, and S65C mutations) were analysed in 250 healthy control subjects and collected retrospectively in 296 patients with suspected iron overload (elevated serum ferritin and/or transferrin saturation). The frequency of patients having at least mild iron overload, and mean serum ferritin and transferrin saturation values were calculated for each HFE genotype. For patients carrying the S65C mutation, clinical data, liver biopsy results, and amount of blood removed at phlebotomy were determined. RESULTS: The HFE S65C mutation was found in 14 patients and eight controls. In controls, the S65C allele frequency was 1.6%. The S65C allele frequency was enriched in non-C282Y non-H63D chromosomes from patients (4.9%) compared with controls (1.9%) (p<0.05). Serum ferritin was significantly increased in controls carrying the S65C mutation compared with those without HFE mutations. Fifty per cent of controls and relatives having the S65C mutation had elevated serum ferritin levels or transferrin saturation. The number of iron overloaded patients was significantly higher among those having HFE S65C compared with those without any HFE mutation. Half of patients carrying the S65C mutation (7/14) had evidence of mild or moderate hepatic iron overload but no signs of extensive fibrosis in liver biopsies. Screening of relatives revealed one S65C homozygote who had no signs of iron overload. Compound heterozygosity with S65C and C282Y or H63D did not significantly increase the risk of iron overload compared with S65C heterozygosity alone. CONCLUSIONS: The HFE S65C mutation may lead to mild to moderate hepatic iron overload but neither clinically manifest haemochromatosis nor iron associated extensive liver fibrosis was encountered in any of the patients carrying this mutation.

Characterization of two novel mutations in the glucocorticoid receptor gene in patients with primary cortisol resistance.

OBJECTIVE: Primary glucocorticoid resistance is characterized by decreased sensitivity to cortisol signalling. We have performed genetic analysis of the glucocorticoid receptor (GR) gene in 12 unrelated patients with primary cortisol resistance as defined by a pathological dexamethasone suppression test. METHODS: Exon specific polymerase chain reaction amplification of the GR gene and sequencing of each exon was carried out. The two mutations were characterized in vitro in terms of glucocorticoid driven reporter gene activity in a transient transfection assay and in a ligand binding assay. Molecular modelling of the R477H mutant was performed based on the X-ray structure of the GR-DNA binding domain. RESULTS: Two novel mutations in the GR gene were found: R477H in the DNA-binding domain which is the first reported mutation in that region of the human GR gene and G679S in the ligand binding domain. The R477H mutation showed no transactivating capacity, whereas the G679S mutation had reduced transactivation capacity compared to the wild-type (wt) GR. When tested for ligand binding capacity, the G679S mutation had 50% binding affinity compared to the wt GR. The effect of the point mutation R477H was deduced by a comparison between the wt structure and the model of the mutant. The wt GR has direct and water mediated contact with the phosphate groups of the glucocorticoid responsive element (GRE) whereas, in the model, the mutation R477H has no contact with the GRE. The G679S mutation is located on the surface of the ligand binding domain, at a distance from the steroid-binding site. A previously reported polymorphism, AAT to AAC at amino acid position 766, was found in four of the patients. CONCLUSIONS: In two of 12 patients with clinical glucocorticoid resistance, mutant forms of GR could be found. The glucocorticoid resistance in vivo in these two patients corresponds to impaired function of the two mutated GR forms in two in vitro assays. The relevance of the conservative polymorphism for the glucocorticoid insensitivity noted in these patients remains to be clarified.

Regulation of the ligand binding activity of the human very low density lipoprotein receptor by protein kinase C-dependent phosphorylation.

The very low density lipoprotein receptor (VLDL-R) binds and internalizes several ligands, including very low density lipoprotein (VLDL), urokinase-type plasminogen activator:plasminogen activator inhibitor type 1 complexes, lipoprotein lipase, and the 39-kDa receptor-associated protein that copurifies with the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor. Although several agonists regulate VLDL-R mRNA and/or protein expression, post-transcriptional regulation of receptor activity has not been described. Here, we report that the ligand binding activity of the VLDL-R in THP-1 monocytic cells, endothelial cells, smooth muscle cells, and VLDL-R-transfected HEK 293 cells is diminished after treatment with phorbol 12-myristate 13-acetate. This response was blocked by inhibitors of protein kinase C (PK-C), including a specific inhibitor of the PK-C beta II isoform, and was associated with phosphorylation of serine residues in the cytoplasmic domain of the receptor. Culture of endothelial cells in the presence of high glucose concentrations, which stimulate diacylglycerol synthesis and PK-C beta II activation, also induced a PK-C-dependent loss of VLDL-R ligand binding activity. Taken together, these studies demonstrate that the ligand binding activity of the VLDL-R is regulated by PK-C-dependent phosphorylation and that hyperglycemia may diminish VLDL-R activity.

The peroxisome proliferator-activated receptor alpha (PPARalpha) regulates bile acid biosynthesis.

Fibrates are a group of hypolipidemic agents that efficiently lower serum triglyceride levels by affecting the expression of many genes involved in lipid metabolism. These effects are exerted via the peroxisome proliferator-activated receptor alpha (PPARalpha). In addition, fibrates also lower serum cholesterol levels, suggesting a possible link between the PPARalpha and cholesterol metabolism. Bile acid formation represents an important pathway for elimination of cholesterol, and the sterol 12alpha-hydroxylase is a branch-point enzyme in the bile acid biosynthetic pathway, which determines the ratio of cholic acid to chenodeoxycholic acid. Treatment of mice for 1 week with the peroxisome proliferator WY-14,643 or fasting for 24 h both induced the sterol 12alpha-hydroxylase mRNA in liver. Using the PPARalpha knockout mouse model, we show that the induction by both treatments was dependent on the PPARalpha. A reporter plasmid containing a putative peroxisome proliferator-response element (PPRE) identified in the rat sterol 12alpha-hydroxylase promoter region was activated by treatment with WY-14,643 in HepG2 cells, being dependent on co-transfection with a PPARalpha expression plasmid. The rat 12alpha-hydroxylase PPRE bound in vitro translated PPARalpha and retinoid X receptor alpha, albeit weakly, in electrophoretic mobility shift assay. Treatment of wild-type mice with WY-14,643 for 1 week resulted in an increased relative amount of cholic acid, an effect that was abolished in the PPARalpha null mice, verifying the functionality of the PPRE in vivo.

The role of O-linked sugars in determining the very low density lipoprotein receptor stability or release from the cell.

The very low density lipoprotein receptor is a member of the low density lipoprotein receptor supergene family for which two isoforms have been reported, one lacking and the other containing an O-linked sugar domain. In order to gain insight into their functionality, transient and stable transformants separately overexpressing previously cloned bovine variants were analyzed. We report evidence that the variant lacking the O-linked sugar domain presented a rapid cleavage from the cell and that a large amino-terminal very low density lipoprotein receptor fragment was released into the culture medium. As only minor proteolysis was involved in the other very low density lipoprotein receptor variant, the clustered O-linked sugar domain may be responsible for blocking the access to the protease-sensitive site(s). To test this hypothesis, a mutant Chinese hamster ovary cell line, ldlD, with a reversible defect in the protein O-glycosylation, was used. The instability of the O-linked sugar-deficient very low density lipoprotein receptor on the cell surface was comparable to that induced by the proteolysis of the variant lacking the O-linked sugar domain. Moreover, our data suggest that the O-linked sugar domain may also protect the very low density lipoprotein receptor against unspecific proteolysis. Taken together, these results indicate that the presence of the O-linked sugar domain may be required for the stable expression of the very low density lipoprotein receptor on the cell surface and its absence may be required for release of the receptor to the extracellular space. The exclusive expression of the variant lacking the O-linked sugar domain in the bovine aortic endothelium opens new perspectives in the physiological significance of the very low density lipoprotein receptor.

Thyroid hormone suppresses hepatic sterol 12alpha-hydroxylase (CYP8B1) activity and messenger ribonucleic acid in rat liver: failure to define known thyroid hormone response elements in the gene.

Sterol 12alpha-hydroxylase (CYP 8B1) is a microsomal cytochrome P450 enzyme involved in bile acid synthesis that is of critical importance for the composition of bile acids formed in the liver. Thyroidectomy of rats caused a more than twofold increase of CYP8B1 and an almost fourfold increase of the corresponding mRNA levels compared to sham-operated rats. Treatment of intact rats with thyroxine caused a 60% reduction of enzyme activity and a 50% reduction of mRNA levels compared to rats injected with saline only. To investigate whether the promoter of the gene contains thyroid hormone response elements, the complete structure of the rat gene was defined. In similarity with the corresponding gene in mouse, rabbit and man, the rat gene was found to lack introns. It had an open reading frame containing 1500 bp corresponding to a protein of 499 amino acid residues. Although thyroid hormone decreased CYP8B1 activity and mRNA in vivo, no hitherto described thyroid hormone response elements were identified 1883 bases upstream of the transcription start site. It is concluded that rat CYP8B1 is regulated by thyroid hormone at the mRNA level. The results are discussed in relation to the structure of the gene coding for the enzyme.

VLDL activation of plasminogen activator inhibitor-1 (PAI-1) expression: involvement of the VLDL receptor.

The potential role of the very low density lipoprotein (VLDL) receptor in mediating VLDL-induced plasminogen activator inhibitor-1 (PAI-1) expression was studied in vitro. Cultured endothelial cells incubated with VLDL showed an increased secretion of PAI-1. This response to VLDL could be completely prevented by the receptor-associated protein (RAP) and partially blocked by rabbit polyclonal anti-VLDL receptor IgG. Furthermore, Chinese hamster ovary (CHO) control cells and cells overexpressing the VLDL receptor were transiently transfected with a PAI-1 promoter-reporter construct and incubated with VLDL. The PAI-1 promoter activity in response to VLDL was significantly higher in the VLDL receptor overexpressing cells compared to the control cells. Addition of RAP completely blocked the VLDL-activated PAI-1 transcription. Electromobility shift assay was performed to investigate whether the enhanced PAI-1 promoter activity seen in the VLDL receptor overexpressing cells in response to VLDL involved induction of the previously described VLDL-inducible factor(s) binding to the -675 to -653 region of the PAI-1 promoter. We found that the binding of the VLDL-inducible factor in VLDL receptor overexpressing cells was markedly enhanced by addition of VLDL as compared to control cells where no increased binding could be seen in response to VLDL. In summary, these results indicate that the VLDL receptor is a strong candidate for mediating VLDL effects on PAI-1 synthesis and secretion in cells expressing this receptor.

Structure and chromosomal assignment of the sterol 12alpha-hydroxylase gene (CYP8B1) in human and mouse: eukaryotic cytochrome P-450 gene devoid of introns.

Sterol 12alpha-hydroxylase (CYP8B1) is a hepatic cytochrome P-450 that controls the ratio of cholic acid over chenodeoxycholic acid in bile and thus controls the solubility of cholesterol. Both the human and the mouse CYP8B1 complementary DNA and gene were cloned and structurally characterized. Surprisingly, the genomic DNA from both species was found to lack introns. The major transcript of the human gene was estimated to be 3950 bp, and the putative promoter region was estimated to be at least 1360 bp. The murine structural gene was found to span approximately 3 kb. By using FISH and radiation hybrid mapping techniques, the human CYP8B1 gene was located to chromosome 3p21.3-p22, whereas FISH mapped the murine counterpart to chromosome 9qF4, a region that is homologous to the third human chromosome. The results from the chromosome mapping and Southern blotting indicated that the gene is present in a single copy. Transcription of the mouse and human CYP8B1 genes was initiated from a position situated 51 and 35 bases, respectively, downstream of a consensus TATA box. A homology of 21% for the promoter regions of mouse and human may indicate differences in transcriptional regulation. Although a potent induction of CYP8B1 mRNA was observed upon starvation of mice, the mechanism behind this effect was not revealed by analysis of the promoter for potential cis-acting elements. In the human promoter, several possible cis-acting regions were identified but none of them could be directly related to bile acid metabolism. After transfection of COS cells with the human coding region, mRNA and enzymatic activity for the 12alpha-hydroxylase were identified. This is the first mammalian cytochrome P-450 gene reported to lack introns. The importance of this structural feature for evolution and gene regulation is discussed.

Lipoprotein receptors in acute myelogenous leukemia: failure to detect increased low-density lipoprotein (LDL) receptor numbers in cell membranes despite increased cellular LDL degradation.

The high-affinity degradation of low-density lipoprotein (LDL) is enhanced 3- to 100-fold in leukemic blood cells from patients with acute myelogenous leukemia (AML), suggesting an increased cellular LDL receptor expression. There are, however, inconsistencies regarding the published properties of LDL receptor regulation in AML cells, and previous data on this are indirect. In the present study the aim was to determine whether the LDL receptor number is increased in AML cells. The LDL receptor number was assayed by ligand blot with rabbit 125I-labeled beta-very-low-density lipoprotein (beta-VLDL) of transferred, SDS-polyacrylamide-gel-electrophoresis-separated AML cell membranes. Samples from 10 patients, six with AML, one with chronic myelogenous leukemia in blast crisis, and three with acute lymphoblastic leukemia, were investigated. The LDL receptor expression was strongly suppressed in all samples to levels lower than that of normal mononuclear cells. This was despite the fact that cells from one patient with AML of M4 subtype had a 50- to 100-fold higher 125I labeled LDL degradation compared with normal cells. Immunoblots with antibodies against gp330/megalin and the LDL-receptor-related protein (LRP) and ligand blot using 125I-labeled 39-kd receptor-associated protein (RAP) could not detect gp330/megalin or VLDL receptors. The LRP was abundant in AML samples of M4 and M5b subtype, as determined from both RAP ligand blot and immunoblot using an LRP-specific antibody. It is concluded that LDL receptors are suppressed in AML cells. It is possible that the high degradation of 125I-labeled LDL present in type M4 and M5 AML cells may involve another lipoprotein receptor.

Bovine aortic endothelial cells express a variant of the very low density lipoprotein receptor that lacks the O-linked sugar domain.

The very low density lipoprotein (VLDL) receptor is a member of the low density lipoprotein supergene family of receptors in which differential splicing of mRNA has been reported. We present several lines of evidence showing that bovine aortic endothelial cells exclusively express a VLDL receptor isoform that lacks the O-linked sugar domain i) Western and receptor-associated protein (RAP) ligand blotting gave a single band of about 99 kDa in membrane extracts of bovine aortic endothelial cells (BAEC). ii) Screening of the BAEC cDNA library with the previously characterized human VLDL receptor cDNA as a probe gave several C-terminal-positive clones; all lacked the 84 nucleotides corresponding to exon 16. Polymerase chain reaction (PCR) confirmed that VLDL receptor cDNA encoding exon 16 was absent from the library. iii) Reverse transcription (RT)-PCR analysis of the BAEC mRNA using a pair of oligonucleotide primers that flank the deletion gave only one band of 136 nt. iv) Semiquantitative RT-PCR analysis showed that only the non-O-glycosylated variant was expressed in BAEC. Cell-binding studies with antibodies against the N-terminal domain showed that the BAEC VLDL receptor is present at the plasma membrane, suggesting that the non-glycosylated variant could be functional. In addition, RT-PCR performed in bovine tissues showed that the variant containing the O-linked sugar domain is preferentially expressed in heart, brain, and skeletal muscle, whereas the non-O-glycosylated spliced variant is found in all tissues analyzed. Taken together these results suggest that the differential splicing of the VLDL receptor is cell- and tissue-specific and that the functions of the receptor could depend on the cell type.

Degradation of urokinase plasminogen activator (UPA) in endometrial stromal cells requires both the UPA receptor and the low-density lipoprotein receptor-related protein/alpha2-macroglobulin receptor.

Primary cultures of human endometrial stromal cells expressed a single class of specific high-affinity binding sites for urokinase plasminogen activator (UPA) with a dissociation constant KD 1.0 nmol/l and saturation at 2.0 nmol/l. Similar binding data and number of free binding sites, about 200 fmol/mg protein, were found for UPA in complex with its inhibitor plasminogen activator inhibitor-1 (PAI-1). These binding data agree with those reported for the specific cell surface receptor for UPA, and stromal cell expression of UPA receptor mRNA was identified in Northern blots. Cell surface-bound UPA was degraded at 37 degrees C. Degradation of complexed UPA was more efficient than that of free UPA. Degradation of free UPA did not require prior binding to endogenous PAI-1. Degradation of both free and complexed UPA was reduced by 70% by colchicine, chloroquine and methylamine, indicating that degradation involved both internalization and lysosomal enzymes. Furthermore, degradation was independently inhibited by about 70% with anti-UPA receptor antibodies and receptor-associated protein, indicating that the UPA receptor as well as one or more receptors of the low-density lipoprotein (LDL) receptor supergene family were involved in the degradation process. Receptor-associated protein ligand blotting demonstrated a major band co-migrating with the LDL receptor-related protein or glycoprotein 330/megalin, and a minor band co-migrating with the very low-density lipoprotein receptor. Immunoblotting positively demonstrated expression of LDL receptor-related protein, but not glycoprotein 330.

Uremic serum enhances scavenger receptor expression and activity in the human monocytic cell line U937.

The macrophage scavenger receptor (SR) plays a leading role in atherogenesis, but little is known about the relevance of SR to atherosclerosis in uremia. In this study, the impact of uremic serum on SR expression and activity was examined in the human monocytic cell line U937. The cells were cultured with serum from ten healthy subjects, ten hemodialysis (HD) and ten continuous ambulatory peritoneal dialysis (CAPD) patients. SR mRNA expression was examined using reverse transcriptase-polymerase chain reaction followed by Southern blot. SR protein amount was evaluated by ligand blot. SR activity was analyzed by cellular uptake of fluorescently labeled acetylated low-density lipoprotein using flow cytometry. Uremic serum dose-dependently enhanced SR activity primarily by increasing the amount of receptor protein. Heat-inactivated uremic serum had a stimulatory effect, but ultrafiltrate of uremic serum, which included molecules with a molecular weight less than ten kDa, had no effect. The serum levels of macrophage-colony stimulating factor (M-CSF), an activator of SR, were fourfold higher in uremia and significantly correlated with SR activity in cells treated with uremic serum. Pre-treatment of uremic serum with a neutralizing antibody to M-CSF abolished the effect of uremic serum on SR activity. In conclusion, uremic serum contains a factor(s) that enhances SR expression and activity in U937 cells. Elevated M-CSF in uremic serum could be responsible for this enhancement.

VLDL receptor mediates the uptake of human chylomicron remnants in vitro.

The VLDL receptor has been described as a new member of the LDL receptor supergene family that specifically binds VLDL in vitro via apolipoprotein E and lipoprotein lipase. Both apolipoprotein E and lipoprotein lipase are constituents of chylomicron remnants, another triglyceride-rich lipoprotein which has been proposed as a physiological ligand for the VLDL receptor. We used human chylomicron remnants to study their uptake into LDL, receptor-deficient Chinese hamster ovary cells overexpressing the human VLDL receptor. The uptake into these cells was compared to that into cells transfected with an empty transfection vector. Human chylomicron remnants were produced in vitro by hydrolysis with lipoprotein lipase, and were labeled with 125I. The uptake of these remnants into the cells overexpressing the VLDL receptor was found to be about 3-fold higher than the uptake into the control cells. The addition of a surplus of either apolipoprotein E or inactivated lipoprotein lipase to the remnants led to an increase in particle uptake. The chylomicron remnant uptake was inhibited by addition of the 39 kDa receptor associated protein These in vitro experiments strongly support the idea that the VLDL receptor is a physiological receptor for chylomicron remnants. The increase of receptor-mediated uptake induced by the addition of apoE or lipoprotein lipase underlines the role of these two proteins in this process.

Expression of very low density lipoprotein receptor in the vascular wall. Analysis of human tissues by in situ hybridization and immunohistochemistry.

The recently cloned very low density lipoprotein (VLDL) receptor binds triglyceride-rich, apolipoprotein-E-containing lipoproteins with high affinity. The observation that VLDL receptor mRNA is abundantly expressed in extracts of tissues such as skeletal muscle and heart, but not liver, has led to the hypothesis that this receptor may facilitate the peripheral uptake of triglyceride-rich lipoproteins. However, little information is available concerning the types of cells that express this receptor in vivo. As expression of the VLDL receptor in the vascular wall might have important implications for the uptake and transport of triglyceride-rich lipoproteins, and perhaps facilitate the development of atherosclerosis in hypertriglyceridemic individuals, we used in situ hybridization and immunohistochemistry to determine whether VLDL receptor mRNA and protein was expressed in human vascular tissue. We observed expression of the receptor by both endothelial and smooth muscle cells within normal arteries and veins, as well as within atherosclerotic plaques. In the latter, the VLDL receptor was also expressed by macrophage-derived foam cells. The widespread distribution of the VLDL receptor in vascular tissue suggests a potentially important role for this receptor in normal and pathophysiological vascular processes.