PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate lipid and glucose metabolism and cellular differentiation. PPAR-alpha and PPAR-gamma are both expressed in human macrophages where they exert anti-inflammatory effects. The activation of PPAR-alpha may promote foam-cell formation by inducing expression of the macrophage scavenger receptor CD36. This prompted us to investigate the influence of different PPAR-activators on cholesterol metabolism and foam-cell formation of human primary and THP-1 macrophages. Here we show that PPAR-alpha and PPAR-gamma activators do not influence acetylated low density lipoprotein-induced foam-cell formation of human macrophages. In contrast, PPAR-alpha and PPAR-gamma activators induce the expression of the gene encoding ABCA1, a transporter that controls apoAI-mediated cholesterol efflux from macrophages. These effects are likely due to enhanced expression of liver-x-receptor alpha, an oxysterol-activated nuclear receptor which induces ABCA1-promoter transcription. Moreover, PPAR-alpha and PPAR-gamma activators increase apoAI-induced cholesterol efflux from normal macrophages. In contrast, PPAR-alpha or PPAR-gamma activation does not influence cholesterol efflux from macrophages isolated from patients with Tangier disease, which is due to a genetic defect in ABCA1. Here we identify a regulatory role for PPAR-alpha and PPAR-gamma in the first steps of the reverse-cholesterol-transport pathway through the activation of ABCA1-mediated cholesterol efflux in human macrophages.

Lipid traffic between high density lipoproteins and Plasmodium falciparum-infected red blood cells.

Several intraerythrocytic growth cycles of Plasmodium falciparum could be achieved in vitro using a serum free medium supplemented only with a human high density lipoprotein (HDL) fraction (d = 1.063-1.210). The parasitemia obtained was similar to that in standard culture medium containing human serum. The parasite development was incomplete with the low density lipoprotein (LDL) fraction and did not occur with the VLDL fraction. The lipid traffic from HDL to the infected erythrocytes was demonstrated by pulse labeling experiments using HDL loaded with either fluorescent NBD-phosphatidylcholine (NBD-PC) or radioactive [3H]palmitoyl-PC. At 37 degrees C, the lipid probes rapidly accumulated in the infected cells. After incubation in HDL medium containing labeled PC, a subsequent incubation in medium with either an excess of native HDL or 20% human serum induced the disappearance of the label from the erythrocyte plasma membrane but not from the intraerythrocytic parasite. Internalization of lipids did not occur at 4 degrees C. The mechanism involved a unidirectional flux of lipids but no endocytosis. The absence of labeling of P. falciparum, with HDL previously [125I]iodinated on their apolipoproteins or with antibodies against the apolipoproteins AI and AII by immunofluorescence and immunoblotting, confirmed that no endocytosis of the HDL was involved. A possible pathway of lipid transport could be a membrane flux since fluorescence videomicroscopy showed numerous organelles labeled with NBD-PC moving between the erythrocyte and the parasitophorous membranes. TLC analysis showed that a partial conversion of the PC to phosphatidylethanolamine was observed in P. falciparum-infected red cells after pulse with [3H]palmitoyl-PC-HDL. The intensity of the lipid traffic was stage dependent with a maximum at the trophozoite and young schizont stages (38th h of the erythrocyte life cycle). We conclude that the HDL fraction appears to be a major lipid source for Plasmodium growth.

Liver X receptor activation controls intracellular cholesterol trafficking and esterification in human macrophages.

Liver X receptors (LXRs) are nuclear receptors that regulate macrophage cholesterol efflux by inducing ATP-binding cassette transporter A1 (ABCA1) and ABCG1/ABCG4 gene expression. The Niemann-Pick C (NPC) proteins NPC1 and NPC2 are located in the late endosome, where they control cholesterol trafficking to the plasma membrane. The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant governing its availability for efflux to extracellular acceptors. Here we investigated the influence of LXR activation on intracellular cholesterol trafficking in primary human macrophages. Synthetic LXR activators increase the amount of free cholesterol in the plasma membrane by inducing NPC1 and NPC2 gene expression. Moreover, ABCA1-dependent cholesterol efflux induced by LXR activators was drastically decreased in the presence of progesterone, which blocks postlysosomal cholesterol trafficking, and reduced when NPC1 and NPC2 mRNA expression was depleted using small interfering RNA. The stimulation of cholesterol mobilization to the plasma membrane by LXRs led to a decrease in cholesteryl ester formation and Acyl-coenzyme A cholesterol acyltransferase-1 activity. These data indicate that LXR activation enhances cholesterol trafficking to the plasma membrane, where it becomes available for efflux, at the expense of esterification, thus contributing to the overall effects of LXR agonists in the control of macrophage cholesterol homeostasis.

Peroxisome proliferator-activated receptor alpha reduces cholesterol esterification in macrophages.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor activated by fatty acid derivatives and hypolipidemic drugs of the fibrate class. PPARalpha is expressed in monocytes, macrophages, and foam cells, suggesting a role for this receptor in macrophage lipid homeostasis with consequences for atherosclerosis development. Recently, it was shown that PPARalpha activation promotes cholesterol efflux from macrophages via induction of the ABCA1 pathway. In the present study, the influence of PPARalpha activators on intracellular cholesterol homeostasis was investigated. In human macrophages and foam cells, treatment with fibrates, synthetic PPARalpha activators, led to a decrease in the cholesteryl ester (CE):free cholesterol (FC) ratio. In these cells, PPARalpha activation reduced cholesterol esterification rates and Acyl-CoA:cholesterol acyltransferase-1 (ACAT1) activity. However, PPARalpha activation did not alter ACAT1 gene expression, whereas mRNA levels of carnitine palmitoyltransferase type 1 (CPT-1), a key enzyme in mitochondrial fatty acid catabolism, were induced. Finally, PPARalpha activation blocked CE formation induced by TNF-alpha, possibly due to the inhibition of neutral sphingomyelinase activation by TNF-alpha. In conclusion, our results identify a role for PPARalpha in the control of cholesterol esterification in macrophages, resulting in an enhanced availability of FC for efflux through the ABCA1 pathway.

Low-density lipoprotein for delivery of an acrylophenone antineoplastic molecule into malignant cells.

In vitro and in vivo data have indicated that tumor cells actively internalize the low-density lipoprotein (LDL) from the circulation. A family of 2-(aminomethyl) acrylophenones (AMA) possesses an in vitro antileukemic activity but is devoid of any in vivo antineoplastic activity, because the compounds are actively captured by proteins in solution in the blood. In order to achieve a selective delivery of these drugs via the LDL pathway, we have incorporated an AMA drug, 2-morpholinomethyl-2',3',4'- trimethoxy acrylophenone hydrochloride (ILE) into LDL particles. ILE spontaneously associated with LDL to produce an LDL-ILE complex containing 200 +/- 100 molecules of drug per LDL particle. The LDL-ILE complex was highly electronegative as detected by electrophoresis. Further, this complex presented an immunologically detected over expression of the ligand-binding domain to the LDL receptor. In spite of these modifications, the LDL receptor processing bound, internalized, and degraded the LDL-ILE complex. Nevertheless, these biological properties were reduced by 32, 20, and 40%, respectively, in comparison to native LDL. Despite its high electronegativity, the LDL-ILE complex was not recognized by the macrophagic scavenger receptor. The LDL-ILE complex showed specific LDL receptor mediated in vitro cytotoxicity as judged from the growth inhibition of neoplastic A549 cells and of normal fibroblasts, but no activity on defective LDL receptor cells. Further, the pharmacological activity of the complex against A549 cells has been demonstrated to be equally potent as that of the free drug (median inhibitory dose, 5 microM). It is suggested that LDL drug targeting of AMA molecules could specifically deliver active molecules to cancer cells, avoiding their entrapment by other blood proteins and their rapid clearance by the reticuloendothelial system.

Immunochemical characterization of two antigenic sites on human apolipoprotein A-I; localization and lipid modulation of these epitopes.

Two monoclonal antibodies, A17 and A30, were raised against human apolipoprotein A-I (apo A-I). They were studied by competitive inhibition of 125I-labeled HDL3 with HDL subfractions, delipidated apo A-I, and complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I and apo A-II. Immunoblotting located the A17 antibody on CNBr fragment 4 of apo A-I and the A30 antibody on CNBr fragment 1. The A17 antigenic determinant was expressed identically in all HDL subclasses, on delipidated apo A-I as well as all on the DMPC-apo A-I and DMPC-apo A-I/apo A-II complexes. In contrast, the apparent affinity constant of the A30 antibody for delipidated apo A-I was about 30-times less than for HDL3 or for apo A-I/apo A-II-phospholipid complexes. These data suggest that the association of apo A-I with phospholipids improves the reactivity of the A30 monoclonal antibody towards apo A-I, and that this antigenic determinant has a different conformation in delipidated apo A-I compared to apo A-I complexed with phospholipids. Turbidimetric and fluorescence experiments monitoring the phospholipid-apo A-I association in the presence and in the absence of the A17 and A30 antibodies were consistent with the competition experiments carried out by solid phase radioimmunoassay (RIA). After reaction of apo A-I with the A30 antibody, we observed an enhancement of the degradation kinetics of large multilamellar vesicles (LMV), while the A17 antibody did not have a significant effect. Calcein leakage experiments carried out below the transition temperature of DPPC showed an enhancement of the degradation kinetics with both monoclonal antibodies, while the phase-transition release was independent of the reaction of apo A-I with the monoclonal antibodies. These data therefore suggest the existence of at least two different types of epitope on apo A-I, which might account for the differences in immunological reactivity of apo A-I that is either delipidated or present on HDL.

Determination of the LDL receptor binding capacity of human lymphocytes by immunocytofluorimetric assay.

The determination of the LDL receptor binding capacity of human blood lymphocytes was assessed by indirect immunocytofluorimetric assay. To produce the maximal synthesis of the LDL receptor, the cholesterol efflux was enhanced by incubation of lymphocytes with HDL3 subfractions. The binding capacity of the LDL receptor was measured by incubation at 4 degrees C either with LDL and rabbit anti-LDL immunoglobulins or with peptide receptor antibody (ARP-Ig) raised against the NH2-terminal sequence of the LDL receptor. Thereafter complexes were incubated with fluorescein-labelled anti-rabbit immunoglobulin (FITC-Ig). Fluorescence flow cytometry was used to quantify the number of fluorescent lymphocytes and results were expressed as the percentage of lymphocytes with a fluorescent intensity above the threshold. Using preimmune rabbit immunoglobulin and then FITC-Ig, only 5-10% of cells were fluorescent. Neither LDL nor ARP-Ig could bind to homozygous familial hypercholesterolemia (FH) lymphocytes. Normal lymphocytes preincubated with HDL3 could bind LDL or ARP-Ig, the number of fluorescent cells being 59 and 39.2% respectively. Subjects with confirmed or suspected heterozygous FH demonstrated cell fluorescence at about half the normal level.

Isolation and characterization of human Lp-B lipoprotein containing apolipoprotein B as the sole apolipoprotein.

A sequential immunoaffinity chromatography procedure was developed to isolate from whole normolipidemic human plasma a subpopulation of apoB containing particles (Lp-B) which is virtually free of non apoB protein. The absence of non apoB protein in Lp-B was assessed by enzyme immunoassay against apolipoproteins A-I, A-II, A-IV, E, C-III and (a). Electron microscopy and fractionation of the isolated particles by gel filtration demonstrated that these particles were heterogeneous in size. However, most of them had diameters between 18 and 26 nm. These particles were found to be rich in cholesterol (molar ratio cholesterol/apoB = 2246 +/- 995) poor in triacylglycerol (molar ratio triacylglycerol/apoB = 555 +/- 518) and had a phospholipids/apoB molar ratio of 713 +/- 348. Most of the cholesterol was esterified (66% +/- 5%). Lp-B particles bound to the apoB, E receptor of HeLa cells with a lower affinity than LDL prepared by ultracentrifugation (1.030 kg/l less than d less than 1.053 kg/l). (KD = 18.9 vs 10.5 nmol/l).

[Purification and characterization of protease inhibitors from urine during pregnancy (author's transl)]. / Purification et caracterisation des inhibiteurs de protéases de l'urine humaine.

Two forms of urinary trypsin inhibitor, A and B, were purified from the pooled urine from pregnant women using non-denaturing methods. The inhibitor B arose from the inhibitor A and was not present in native urine. Electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate indicated a new heterogeneity of the inhibitor B with molecular weights of 33 000 and 24 000; the molecular weight obtained for the inhibitor A was 50 000. Inhibitors A and B were acidic proteins with an isoelectric pH of about 2.6 for A and about 4.2 for B. Inhibitor A and inter-alpha-trypsin inhibitor formed a precipitate with an antiserum to purified inhibitor B. But neither inhibitor A nor inhibitor B formed a precipitate with anti whole human serum or anti-inter-alpha-trypsin inhibitor antiserum. Measurements of specific activity of inhibitor A were consistent with two active sites in the molecule.

Isolation and characterization of two sub-species of Lp(a), one containing apo E and one free of apo E.

Lipoprotein Lp(a) was isolated by immunoaffinity chromatography using anti apolipoprotein B and anti apolipoprotein (a) immunosorbents. Besides apolipoproteins (a) and B, this fraction was shown to contain apolipoproteins C and E. Therefore, it was decided to further purify this crude Lp(a) into particles containing apolipoprotein E and particles free of apo E, using chromatography with an anti apolipoprotein E immunosorbent. Lp(a), free of apolipoprotein E was cholesterol ester rich and triacylglycerol poor and was found mainly in the LDL size range. In contrast, Lp(a) containing apolipoprotein E was triacylglycerol rich and was distributed mainly in the VLDL and IDL size range. Binding of these two fractions, one containing apo E and one free of it, to the apo B/E receptor of HeLa cells was studied. Both fractions bound to the receptor but the one containing apo E had a better affinity than the one free of apo E. Further studies are needed to identify the clinical importance of these two different entities.

HDL3 binds to glycosylphosphatidylinositol-anchored proteins to activate signalling pathways.

Previous studies have indicated that in HepG2 cells HDL3-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins. HDL3-binding to HepG2 cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL3 binds directly on GPI-anchored proteins to initiate signaling. Moreover HDL3-binding was found to be partly inhibited by antibodies against the HDL-binding protein (AbHBP). HDL3, when binding to HepG2 cells, promoted the release in the culture medium of a 110 kDa protein that binds AbHBP, while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (AbIPG), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL3-induced release of the 110 kDa protein in the culture medium. In [3H]-PC prelabeled HepG2 cells, AbHBP were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL3 stimulation. Cellular preincubation with AbIPG was found to inhibit only the HDL3-signal and not the AbHBP-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL3-and AbHBP-signal. Moreover cellular preincubation with AbHBP for 1 h at 37 degrees C was found to inhibit HDL3-signalling pathways. Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL3-signalling pathways as binding sites.

Structural domain of apolipoprotein A-I involved in its interaction with cells.

Apolipoprotein A-I (apo A-I) is the major protein constituent of high-density lipoprotein (HDL), the lipoprotein fraction which mediates the reverse cholesterol transport. This apolipoprotein plays an important role in the binding of HDL to cells and participates in the efflux of cellular cholesterol. We have recently compared six different genetic variants of apo A-I and found that the apo A-I (Pro 165-->Arg) mutant is defective in promoting cellular cholesterol efflux from murine adipocytes and peritoneal macrophages and we have proposed that this region of apo A-I may be involved in their interaction with cells. To confirm this hypothesis, four monoclonal antibodies (mAbs) specific for apo A-I were used to study the inhibition of the interaction of palmitoyloleoylphosphatidylcholine (POPC): apoA-I complexes with HeLa cells and adipocytes. Among these antibodies, the apo A-I epitope recognized by the A44 mAb lies in the COOH terminal region (amino acid residues 149-186) including the proposed region. The antibodies A05, and A03 react with residues 25-82, 135-140, respectively and the A11 mAb corresponds to a discontinuous epitope at residues 99-105 and 126-132. Our results show clearly that the A44 and A05 mAbs reduce both the binding to HeLa cells and the cholesterol efflux from adipocytes. The inhibition of POPC: apoA-I complexes binding to both cell types is more strictly observed with the Fab fragments of monoclonal antibodies A44 and A05. Partial cotitration curves of these mAbs in a solid phase assay (RIA), indicated partial competition between these two antibodies. We propose a structural model for the POPC: apoA-I complexes where the N-terminal domain of one apo A-I molecule is in close spatial relationship with the C-terminal domain of the adjacent apo A-I molecule. We therefore suggest that the domain around amino acid 165 of apo A-I and which is recognized by mAb A44 (149-186) forms or contains some specific regions which mediate selectively the interaction with the binding site of cells and is involved in the efflux of cellular cholesterol.

Lipoprotein pattern and plasma lecithin cholesterol acyl transferase activity in children with Alagille syndrome.

Alagille syndrome is frequently associated with hyperlipidemia and xanthoma. The aim of the study was to assess the lipid profile (plasma lipoproteins, apolipoproteins (apo)) and lecithin cholesterol acyl transferase (LCAT) activity, with and without treatment with cholestyramine in Alagille syndrome. Five children (mean age = 6 +/- 4 years) with Alagille syndrome were studied at two different times while receiving no treatment, and while receiving cholestyramine. They were compared with 12 normal controls, who were not different from patients for age and sex. In Alagille syndrome, total serum cholesterol, triglycerides and phospholipids were elevated compared with the controls (P < 0.008). VLDL-cholesterol, LDL-cholesterol, HDL-triglycerides, LDL-triglycerides and VLDL-phospholipids were higher, whereas HDL-cholesterol was lower than controls (P < 0.03). Apo B, CIII, E and lipoprotein particles Lp AI were higher (P < 0.001), whereas Lp AI:AII was lower than controls (P < 0.03). Lipoprotein-X was present in the 5 children with Alagille syndrome and explained in part the elevation of plasma cholesterol, phospholipids, and apo CIII. LCAT activity was decreased (P < 0.01) and might cause some abnormalities of HDL with lower cholesterol, higher triglycerides, apo E and apo CIII contents than controls, and abnormalities of VLDL and LDL with higher cholesterol, triglycerides, phospholipids and apo B contents than controls. Some of the risk factors of atherosclerosis were found in Alagille syndrome, namely high levels of plasma cholesterol, LDL cholesterol, apo B, apo B/apo AI. Treatment with cholestyramine resulted in a few modifications to the lipid profile, while lipoprotein-X and the decrease of LCAT activity persisted.

Effects of fenofibrate, gemfibrozil and nicotinic acid on plasma lipoprotein levels in normal and hyperlipidemic mice. A proposed model for drug screening.

These studies examine the effects of a hypercholesterolemic diet (with butter and cholesterol added), or a hypertriglyceridemic diet (30% sucrose (w/v) in drinking water), on murine plasma lipids and lipoproteins prepared either by sequential ultracentrifugation or gel exclusion chromatography. The hypercholesterolemic diet increased plasma cholesterol (186%), particularly that associated with very low (VLDL) and low (LDL) density lipoproteins. In addition, the cholesterol/triglyceride ratio in the VLDL fraction rose significantly from 0.10 to 4.0. The hypertriglyceridemic diet raised markedly plasma triglyceride levels (46%) by expanding the circulating VLDL pool (+39%). These dietary modifications were used to provide a model for the examination of 3 classical hypolipidemic drugs (fenofibrate, gemfibrozil and nicotinic acid). In animals receiving the standard diet, fenofibrate, gemfibrozil and nicotinic acid decreased the triglyceride (TG) level (-28%, -31%, and -38%) by lowering VLDL-TG (-37%, -42%, and -49%), fenofibrate and gemfibrozil increased HDL-cholesterol by 18% and 31%, respectively. In animals receiving the hypercholesterolemic diet fenofibrate lowered the total plasma cholesterol level by 40%, at the same time increasing HDL-cholesterol by 23%. In animals fed sucrose, on the other hand, fenofibrate (100 mg/kg per day) and nicotinic acid (900 and 600 mg/kg per day) reduced plasma triglyceride levels (-20%, -40% and -33%), and nicotinic acid (900 mg/kg per day) decreased VLDL-TG by 58%. These results are in good agreement with clinical data from studies in man and suggest that this animal model may provide a useful and rapid screening system for testing new lipid lowering drugs.

Effects of fenofibrate on lipoprotein metabolism and fatty acid distribution in Zucker rats.

The short term effects of fenofibrate (150 mg/kg per day), administered by gavage, on lipoprotein and fatty acid distribution have been investigated in an hypertriglyceridemic model, the Zucker rat. Lean rats were compared to control obese and treated obese rats, and control obese animals to treated obese littermates. Classically, plasma cholesterol and triacylglycerol increased by 1.8- and 7.9-fold, respectively, in control obese versus lean rats. Treatment of the Zucker obese rats with fenofibrate reduced their plasma cholesterol by 10% and raised triacylglycerol by 47% (P less than 0.001) in comparison to untreated control obese rats. These effects were accompanied by a change in the composition of all plasma lipoproteins. The cholesterol/triacylglycerol ratio in VLDL rose by 32% while that in LDL and HDL fell by 43 and 47%, respectively. Drug therapy altered the fatty acid profile in both plasma and liver; the percentage of polyunsaturated fatty acids fell while monounsaturated fatty acids increased. The increased proportion of monounsaturated fatty acids in plasma suggests that the fatty acid composition of circulating lipoproteins is modified, particularly in VLDL. This, in association with the altered lipid distribution in VLDL may reflect an abnormal metabolism of this lipoprotein. In view of these abnormalities, we conclude that this rat is not an appropriate model for the short-term study of clofibrate analogues.

Apo B-containing lipoprotein particles in poorly controlled insulin-dependent diabetes.

The goal of this study was to compare the structural and biological characteristics of apolipoprotein (apo) B-100-containing particle subfractions isolated from poorly controlled diabetic patients with insulin-dependent diabetes (IDDM), and healthy controls matched for sex, age and body mass index (BMI). Different apo B-containing particles were isolated by sequential immunochromatography and were free of apo A-I, apo A-II, apo A-IV and apo(a). Particles lipoprotein (Lp) B/C-III contained apo B and apo C-III. They were free of apo E. Particles Lp B/E contained apo B and apo E. They were free of apo C-III. Particles Lp B were devoided of apo C-III and apo E. All these particles could contain other known apolipoproteins not cited here, as for example apo C-II and/or apo C-I. The plasma levels of cholesterol, triglycerides, phospholipids, apo A-I, B-100, C-III, E, total Lp B/C-III, total Lp B/E were not different between patients and controls. The physico-chemical properties of Lp B/C-III and Lp B/E were similar in both groups. Only Lp B from patients exhibited some changes, an increase in the size and a decrease in the cholesterol and cholesteryl ester levels. The conformational properties of the lipoproteins were studied through their immunoreactivity against four different anti-apo B-100 monoclonal antibodies (MAb) for which sequential epitopes have been located on the protein, and one MAb for which the epitope is conformationally expressed. Again, minor changes were observed between patients and controls, and only a slight decrease in the immunoreactivity of the epitope encompassing amino-acid residues 405 to 539 of Lp B and of the conformationally expressed epitope of Lp B/C-III were found in patients. Nevertheless, whatever these conformational and/or physico-chemical modifications may be, they were not sufficient to induce functional alterations in the binding of the particles from the patients to the LDL-receptor of HeLa cells. This study shows that IDDM is not associated with any significant abnormalities in the apo-containing lipoprotein particles. The excessive occurrence of coronary heart disease (CHD) and other atherosclerotic vascular disease in patients with IDDM must have other causes.

Branched synthetic peptide constructs mimic cellular binding and efflux of apolipoprotein AI in reconstituted high density lipoproteins.

This study investigates the suitability of the trimeric apolipoprotein (apo)AI(145-183) peptide that we recently described, to serve as a model to probe the relationship between apoAI structure and function. Three copies of the apoAI(145-183) unit, composed each of two amphipathic alpha-helical segments, were branched onto a covalent core matrix and the construct was recombined with phospholipids. A similar construct was made with the apoAI(102-140) peptide and used as a comparison with dimyristoylglycerophosphocholine (DMPC)-apoAI complexes. The DMPC-trimeric-apoAI(145-183) complexes had similar immunological reactivity with monoclonal antibodies directed against the 149-186 apoAI sequence (A44), suggesting that the A44 epitope is exposed similarly in both the synthetic peptide and the native apoAI complexes. The complexes generated with the trimeric-apoAI(145-183) bind specifically to HeLa cells with comparable affinity to the DMPC apoAI complexes; they are a good competitor for binding of apoAI to both HeLa cells and Fu5AH rat hepatoma cells; finally, these complexes promote cholesterol efflux from Fu5AH cells with an efficiency comparable with the apo AI/lipid complexes. To study LCAT activation by the trimeric apo AI(145-183) construct, complexes were prepared with dipalmitoylphosphatidylcholine (DPPC), cholesterol (C) and either the trimeric construct or apoAI. LCAT activation by the trimeric construct was much lower than by apo AI, possibly because the conformation of the trimeric 145-183 peptide in DPPC/C/peptide complexes does not mimic that of apoAI in the corresponding complexes. In comparison, the complexes generated with the multimeric apoAI(102-140) construct had a poor capacity to mimic the physico-chemical and biological properties of apoAI. The apoAI(102-140) construct had low affinity for lipid compared with the (145-183) construct. After association with lipids, it was a poor competitor of DMPC-apoAI complexes for cellular binding and had only limited capacity to promote cholesterol efflux. These results suggest trimeric constructs can serve as an appropriate models for apoAI, enabling further investigations and new experimental approaches to determine the structure-function relationship of apoAI.

Fh-Souassi: a founder frameshift mutation in exon 10 of the LDL-receptor gene, associated with a mild phenotype in Tunisian families.

Familial hypercholesterolemia (FH) has a higher prevalence in central Tunisia together with a milder clinical expression than in western countries. The molecular basis of FH in Tunisia remains unknown. Our aim was to identify FH-causing mutations in three unrelated families (21 subjects) from the area of Souassi (central Tunisia). In probands with a presentation of homozygous FH, the promoter and 18 exons of the low density lipoprotein (LDL)-receptor gene were sequenced in both orientations. A novel complex frameshift mutation was identified in exon 10, nucleotides 1477-1479 (TCT) at Serine 472 were replaced by an insertion of seven nucleotides (AGAGACA), producing a premature termination codon 43 amino acids downstream. Binding of 125I-labelled LDL at 4 degrees C to cultured fibroblasts from two probands showed <2% normal LDL-receptor activity. AvaII digestion of PCR amplified genomic DNA identified this unique mutation in all families; homozygotes n=11, heterozygotes n=10. All mutation carriers shared the same haplotype (7 RFLPs), suggesting that they had a common ancestor. Despite high plasma LDL levels (m=16.0+/-3.0 mmol/l) and extravascular cholesterol deposits, most homozygotes were diagnosed after puberty and had a delayed onset of cardiovascular complications. Moreover, most heterozygotes were free of clinical signs and had plasma LDL cholesterol in the normal range (4.7+/-1.3 mmol/l) without taking any lipid-lowering medication. This mild clinical phenotype which contrasted with the severity of the mutation, could not be explained by specific apolipoprotein E or lipoprotein lipase alleles.

Lipoproteins containing apolipoprotein B isolated from patients with abetalipoproteinemia and homozygous hypobetalipoproteinemia: identification and characterization.

Abetalipoproteinemia (ABL) and homozygous hypobetalipoproteinemia (HBL) are inherited disorders which are classically characterized by progressive retinal and spinocerebellar disease, fat-soluble vitamin deficiency, and absence of apolipoprotein (apo) B from the plasma. Using immunoaffinity chromatography with an anti-apo B antiserum, we isolated apo B-containing lipoprotein (LpB) particles from the plasma of 4 ABL and 2 HBL patients. The LpB particles were characterized and compared with low density lipoprotein (LDL) and LpB isolated from normal plasma. The ABL/HBL LpB particles were similar in size and charge to normal LpB particles but were relatively enriched in several other apolipoproteins. They contained alpha-tocopherol in a ratio to cholesterol that was proportionately much higher than the very low ratio of alpha-tocopherol to cholesterol in plasma. They bound saturably to fibroblasts and were internalized and degraded similarly to LDL. Hence, the molecular defects in ABL and HBL permit the secretion of a very small number of apo B-containing lipoproteins which may be important for transport of alpha-tocopherol to peripheral tissues.

Lack of toxic effects of F 12511, a novel potent inhibitor of acyl-coenzyme A: cholesterol O-acyltransferase, on human adrenocortical cells in culture.

Inhibition of acyl-coenzyme A: cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) reduces intracellular cholesteryl esters that are substrates for steroidogenesis in adrenal cells. The adrenal side effects of ACAT inhibitors remain a key point for their development as antiatherosclerotic agents. The aim of this study was to characterize the effects of a novel and powerful ACAT inhibitor, F 12511 (S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-phenylacetanilide, on the NCI-H295R cell line, which has functional properties comparable to those of normal human adrenal cells. F 12511 incubated with cultured cells for 4-72 hr strongly inhibited cholesteryl oleate formation. The concentrations required to produce 50% inhibition (IC50) values) ranged from 20 to 50 nM; in the presence of low-density lipoproteins (LDL), this effect was paralleled by a decrease in cholesteryl ester mass and an increase in intracellular free cholesterol. At concentrations 100-fold larger than the IC(50) value for up to 48 hr, F 12511 reduced neither the basal release of cortisol and aldosterone nor the production of cortisol stimulated by forskolin. F 12511 did not modify the mRNA levels of the steroidogenic enzyme genes cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), or cytochrome P450 21-hydroxylase (P450c21) or those of the LDL receptor and high-density lipoprotein scavenger receptor class B, type I (SR-BI) genes, either in the presence or absence of adenosine 3',5'-cyclic monophosphate stimulation for 24 hr. Exposure to F 12511 at up to 3 microM for 24 or 48 hr did not result in significant change in morphological and ultrastructural characteristics; the cytoplasm contained large numbers of mitochondria with intact crystae, and the same typical features of secretory activity were observed in NCI-H295R control cells. Exposure to 3 microM of F 12511 for 96 hr also did not affect cell viability. These data demonstrate that reduction of the substrate for steroidogenesis by the ACAT inhibitor F 12511 impairs neither steroid production nor transcription of genes involved in steroidogenesis and lipoprotein uptake in the pluripotent human adrenal cell line NCI-H295R.