REDD2 gene is upregulated by modified LDL or hypoxia and mediates human macrophage cell death.

OBJECTIVE: Cholesterol accumulation in macrophages is known to alter macrophage biology. In this article we studied the impact of macrophage cholesterol loading on gene expression and identified a novel gene implicated in cell death. METHODS AND RESULTS: The regulated in development and DNA damage response 2 (REDD2) gene was strongly upregulated as THP-1 macrophages are converted to foam cells. These results were confirmed by Northern blot of RNA from human monocyte-derived macrophages (HMDM) treated with oxidized LDL (oxLDL). Human REDD2 shares 86% amino acid sequence identity with murine RTP801-like protein, which is 33% identical to RTP801, a hypoxia-inducible factor 1-responsive gene involved in apoptosis. Treatment of HMDM with desferrioxamine, a molecule that mimics the effect of hypoxia, increased expression of REDD2 in a concentration-dependent fashion. Transfection of U-937 and HMEC cells with a REDD2 expression vector increased the sensitivity of the cells for oxLDL-induced cytotoxicity, by inducing a shift from apoptosis toward necrosis. In contrast, suppression of mRNA expression using siRNA approach resulted in increased resistance to oxLDL treatment. CONCLUSIONS: We showed that stimulation of REDD2 expression in macrophages increases oxLDL-induced cell death, suggesting that REDD2 gene might play an important role in arterial pathology.

Systemic tissue inhibitor of metalloproteinase-1 gene delivery reduces neointimal hyperplasia in balloon-injured rat carotid artery.

Metalloproteinases (MMP)-2 and MMP-9 play a role in smooth muscle cell (SMC) migration from the media to the intima following arterial injury. Intravenous administration of adenovirus encoding tissue inhibitor of metalloproteinase-1 (TIMP-1) into balloon-injured rat arteries (3 x 10(11) viral particles/rat; n=7) resulted in a transient expression of TIMP-1 and a significant inhibition of neointima thickening within 16 days ( approximately 40% vs. control; P=0.012). Three days after injury, the number of intimal SMCs was decreased by approximately 98% in TIMP-1-treated rats. However, no alteration was seen in intimal SMC proliferation after 13 days of injury. Therefore, our results show that systemic gene transfer of TIMP-1 is a promising approach in early restenosis treatment.

Effect of mutations of N- and C-terminal charged residues on the activity of LCAT.

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the alpha/beta hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribution of the N- and C-terminal conserved basic residues to the catalytic activity of this enzyme. Most basic, and some acidic residues, conserved among LCAT proteins from different species, were mutated in the N-terminal (residues 1;-210) and C-terminal (residues 211;-416) regions of LCAT. Measurements of LCAT-specific activity on a monomeric substrate, on low density lipoprotein (LDL), and on reconstituted high density lipoprotein (rHDL) showed that mutations of N-terminal conserved basic residues affect LCAT activity more than those in the C-terminal region. This agrees with the highest conservation of the alpha/beta hydrolase fold and structural homology with pancreatic lipase observed for the N-terminal region, and with the location of most of the natural mutants reported for human LCAT. The structural homology between LCAT and pancreatic lipase further suggests that residues R80, R147, and D145 of LCAT might correspond to residues R37, K107, and D105 of pancreatic lipase, which form the salt bridges D105-K107 and D105-R37. Natural and engineered mutations at residues R80, D145, and R147 of LCAT are accompanied by a substantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme.

Increased levels of high-density lipoprotein cholesterol are ineffective in inhibiting the development of immune responses to oxidized low-density lipoprotein and atherosclerosis in transgenic rabbits expressing human apolipoprotein (apo) A-I with severe hypercholesterolaemia.

High levels of high-density lipoprotein (HDL) cholesterol have been reported to protect against the development of atherosclerosis in humans by increasing reverse cholesterol transport and inhibiting the oxidation of low-density lipoprotein (LDL) due to the paraoxonase content of HDL. The purpose of the present study was to assess if there are any relationships between in vivo increases in serum levels of immunological LDL oxidation markers [autoantibodies against oxidized LDL, autoantibodies against malondialdehyde-modified LDL, LDL immune complexes and anti-cardiolipin autoantibodies], paraoxonase activity and the development of atherosclerosis in control rabbits and in transgenic rabbits expressing human apolipoprotein (apo) A-I. A total of 13 apo A-I transgenic rabbits and 18 non-transgenic littermates were fed on a cholesterol-rich diet (0.4%, w/w) for 14 weeks, and were monitored at weeks 0, 2, 6, 10 and 14. Aortic atherosclerotic lesions were measured at the end of this period. Human apo A-I transgenic rabbits with high HDL cholesterol levels were not protected against the development of atherosclerosis when they were fed on a cholesterol-rich diet which induced dramatic hypercholesterolaemia. Immunological markers of LDL oxidation increased and serum paraoxonase activity decreased similarly in control and transgenic rabbits. In conclusion, the present study demonstrates that high HDL cholesterol levels are ineffective in inhibiting increases in immunological markers of LDL oxidation and the development of atherosclerosis in a mammal with severe hypercholesterolaemia.

Apolipoprotein specificity for lipid efflux by the human ABCAI transporter.

ABCAI, a member of the ATP binding cassette family, mediates the efflux of excess cellular lipid to HDL and is defective in Tangier disease. The apolipoprotein acceptor specificity for lipid efflux by ABCAI was examined in stably transfected Hela cells, expressing a human ABCAI-GFP fusion protein. ApoA-I and all of the other exchangeable apolipoproteins tested (apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, apoE) showed greater than a threefold increase in cholesterol and phospholipid efflux from ABCAI-GFP transfected cells compared to control cells. Expression of ABCAI in Hela cells also resulted in a marked increase in specific binding of both apoA-I (Kd = 0.60 microg/mL) and apoA-II (Kd = 0.58 microg/mL) to a common binding site. In summary, ABCAI-mediated cellular binding of apolipoproteins and lipid efflux is not specific for only apoA-I but can also occur with other apolipoproteins that contain multiple amphipathic helical domains.

Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase.

Previous studies have suggested that the helical repeat formed by residues 143;-164 of apolipoprotein A-I (apoA-I) contributes to lecithin:cholesterol acyltransferase (LCAT) activation. To identify specific polar residues involved in this process, we examined residue conservation and topology of apoA-I from all known species. We observed that the hydrophobic/hydrophilic interface of helix 143;-164 contains a cluster of three strictly conserved arginine residues (R149, R153, and R160), and that these residues create the only significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a series of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Mutation of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipid-protein complexes, whereas control mutations (E146Q, D150N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55%. The markedly decreased activities of mutants R149, R153, and R160 resulted from a decrease in the maximal reaction velocity V(max) because the apparent Michaelis-Menten constant K(m) values were similar for the mutant and wild-type apoA-I proteins. These data suggest that R149, R153, and R160 participate in apoA-I-mediated activation of LCAT, and support the "belt" model for discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointing toward the surface of the disc, enabling interactions with LCAT. - Roosbeek, S., B. Vanloo, N. Duverger, H. Caster, J. Breyne, I. De Beun, H. Patel, J. Vandekerckhove, C. Shoulders, M. Rosseneu, and F. Peelman. Three arginine residues in apolipoportein A-I are critical for activation of lecithin:cholesterol acyltransferase J. Lipid Res. 2001. 42: 31;-40.

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.

Decreased susceptibility to diet-induced atherosclerosis in human apolipoprotein A-II transgenic mice.

Studies performed in vivo have been controversial regarding the implication of human apolipoprotein (apo)A-II in the atherogenic process. Expression of human apoA-II in transgenic mice fed a chow diet leads to (1) a bimodal distribution of high density lipoprotein (HDL) size as in humans, (2) a reduction in total cholesterol concentration that is mainly due to a reduction in non-HDL cholesterol level, and (3) a dramatic reduction in mouse endogenous apoA-I and apoA-II. After 20 weeks on an atherogenic diet, transgenic mice had reduced total cholesterol concentrations because of a reduction in cholesterol associated with all lipoprotein classes. Endogenous apoA-I and apoA-II were also dramatically decreased in transgenic mice. The mean area of atherosclerotic lesions was drastically decreased in transgenic mice (-44%, P=0.0027) compared with control mice. The amount of aortic surface covered by lesions was positively correlated with very low density lipoprotein cholesterol (P<0.01) and intermediate density lipoprotein cholesterol levels (P<0.05). Transgenic mice were protected against the development of atherosclerosis despite a marked decrease in HDL cholesterol and apoA-I concentrations. This protection may be related to the marked reduction in circulating low density lipoprotein (very low density and intermediate density lipoprotein) levels in transgenic mice.

Expression of human apolipoprotein A-I/C-III/A-IV gene cluster in mice induces hyperlipidemia but reduces atherogenesis.

The apolipoprotein (apo)A-I/C-III/A-IV gene cluster is involved in lipid metabolism and atherosclerosis. Overexpression of apoC-III in mice causes hypertriglyceridemia and induces atherogenesis, whereas overexpression of apoA-I or apoA-IV increases cholesterol in plasma high density lipoprotein (HDL) and protects against atherosclerosis. Each gene has been studied alone in transgenic mice but not in combination as the entire cluster. To determine which phenotype is produced by the expression of the entire gene cluster, transgenic mice were generated with a 33-kb human DNA fragment. The results showed that the transgene contained the necessary elements to direct hepatic and intestinal expression of the 3 genes. In the pooled data, plasma concentrations were 257+/-9, 7.1+/-0.5, and 1.0+/-0.2 mg/dL for human apoA-I, apoC-III, and apoA-IV, respectively (mean+/-SEM). Concentrations of these apolipoproteins were higher in males than in females. Human apoA-I and apoC-III concentrations were positively correlated, suggesting that they are coregulated. Transgenic mice exhibited gross hypertriglyceridemia and accumulation of apoB(48)-containing triglyceride-rich lipoproteins. Plasma triglyceride and cholesterol concentrations were correlated positively with human apoC-III concentration, and HDL cholesterol was correlated with apoA-I concentration. In an apoE-deficient background, despite being markedly hypertriglyceridemic, cluster transgenic animals compared with nontransgenic animals showed a 61% reduction in atherosclerosis. This suggests that apoA-I and/or apoA-IV can protect against atherosclerosis even in the presence of severe hyperlipidemia. These mice provide a new model for studies of the regulation of the 3 human genes in combination.

Antiangiogenic effect of interleukin-10 in ischemia-induced angiogenesis in mice hindlimb.

Ischemia induces both hypoxia and inflammation that trigger angiogenesis. The inflammatory reaction is modulated by production of anti-inflammatory cytokines. This study examined the potential role of a major anti-inflammatory cytokine, interleukin (IL)-10, on angiogenesis in a model of surgically induced hindlimb ischemia. Ischemia was produced by artery femoral occlusion in both C57BL/6J IL-10(+/+) and IL-10(-/-) mice. After 28 days, angiogenesis was quantified by microangiography, capillary, and arteriole density measurement and laser Doppler perfusion imaging. The protein levels of IL-10 and vascular endothelial growth factor (VEGF) were determined by Western blot analysis in hindlimbs. IL-10 was markedly expressed in the ischemic hindlimb of IL-10(+/+) mice. Angiogenesis in the ischemic hindlimb was significantly increased in IL-10(-/-) compared with IL-10(+/+) mice. Indeed, angiographic data showed that vessel density in the ischemic leg was 10.2+/-0.1% and 5.7+/-0.4% in IL-10(-/-) and IL-10(+/+) mice, respectively (P:<0.01). This corresponded to improved ischemic/nonischemic leg perfusion ratio by 1.4-fold in IL-10(-/-) mice compared with IL-10(+/+) mice (0.87+/-0. 05 versus 0.63+/-0.01, respectively; P:<0.01). Revascularization was associated with a 1.8-fold increase in tissue VEGF protein level in IL-10(-/-) mice compared with IL-10(+/+) mice (P:<0.01). In vivo electrotransfer of murine IL-10 cDNA in IL-10(-/-) mice significantly inhibited both the angiogenic process and the rise in VEGF protein level observed in IL-10(-/-) mice. No changes in vessel density or VEGF content were observed in the nonischemic hindlimb. These findings underscore the antiangiogenic effect of IL-10 associated with the downregulation of VEGF expression and suggest a role for the inflammatory balance in the modulation of ischemia-induced angiogenesis.

High-level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle.

Numerous diseases are linked to the absence or insufficient concentration of a specific plasma protein. Gene transfer is an appealing strategy for correction of such diseases. We report high and sustained plasma secretion of human secreted alkaline phosphatase and of human Factor IX by skeletal muscle of mice. This was obtained by delivering square-wave unipolar electric pulses of low field strength (200 V/cm) and long duration (20 ms) to skeletal muscle previously injected with plasmid DNA encoding for the secreted protein. This intramuscular electrotransfer method allows 30- to 150-fold increase in reporter protein secretion, compared to simple plasmid DNA injection. This increase allows one to obtain values of up to 2200 ng/ml of a reporter circulating protein. Moreover, this high level of secretion remains stable for several months.

Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter.

The ABCA1 gene, a member of the ATP-binding cassette A (ABCA1) transporter superfamily, encodes a membrane protein that facilitates the cellular efflux of cholesterol and phospholipids. Mutations in ABCA1 lead to familial high density lipoprotein deficiency and Tangier disease. We report the complete human ABCA1 gene sequence, including 1,453 bp of the promoter, 146,581 bp of introns and exons, and 1 kb of the 3' flanking region. The ABCA1 gene spans 149 kb and comprises 50 exons. Sixty-two repetitive Alu sequences were identified in introns 1-49. The transcription start site is 315 bp upstream of a newly identified initiation methionine codon and encodes an ORF of 6,783 bp. Thus, the ABCA1 protein is comprised of 2,261 aa. Analysis of the 1,453 bp 5' upstream of the transcriptional start site reveals multiple binding sites for transcription factors with roles in lipid metabolism. Comparative analysis of the mouse and human ABCA1 promoter sequences identified specific regulatory elements, which are evolutionarily conserved. The human ABCA1 promoter fragment -200 to -80 bp that contains binding motifs for SP1, SP3, E-box, and AP1 modulates cellular cholesterol and cAMP regulation of ABCA1 gene expression. These combined findings provide insights into ABCA1-mediated regulation of cellular cholesterol metabolism and will facilitate the identification of new pharmacologic agents for the treatment of atherosclerosis in humans.

Therapeutic liver repopulation in a mouse model of hypercholesterolemia.

Liver repopulation constitutes an attractive approach for the treatment of liver disorders or of diseases requiring abundant secretion of an active protein. We have described previously a model of selective repopulation of a normal liver by Fas/CD95-resistant hepatocytes, in which we achieved up to 16% hepatocyte repopulation. In the present study, we investigated the therapeutic efficacy of this strategy. With this aim, apolipoprotein E (ApoE) knockout mice were transplanted with Fas/CD95-resistant hepatocytes which constitutively express ApoE. Transplanted mice were submitted to weekly injections of non-lethal doses of the Fas agonist antibody Jo2. After 8 weeks of treatment, we obtained up to 30% of the normal level of plasma ApoE. ApoE secretion was accompanied by a drastic and significant decrease in total plasma cholesterol, which even fell to normal levels. Moreover, this secretion was sufficient to markedly reduce the progression of atherosclerosis. These results demonstrate the efficacy of this repopulation approach for correcting a deficiency in a protein secreted by the liver.

Human ApoA-IV overexpression in transgenic mice induces cAMP-stimulated cholesterol efflux from J774 macrophages to whole serum.

The role of apolipoprotein A-IV (apoA-IV) in lipoprotein metabolism has not been established. The aim of the present study was to investigate the role of apoA-IV in reverse cholesterol transport by comparing cellular cholesterol efflux to serum or serum fractions from control mice and from mice transgenic for human apoA-IV (HuA-IVTg mice). When Fu5AH hepatoma cells were used, the cholesterol efflux to serum from either control or transgenic mice was similar. When control J774 macrophage cells were used, a comparison of efflux to serum or lipoprotein-deficient serum (LPDS) failed to demonstrate any differences between control and transgenic mice. In contrast, when the J774 cells were pretreated with cAMP, there was a stimulation of efflux to whole serum or LPDS from HuA-IVTg mice. cAMP treatment had no effect on efflux to serum or LPDS from control mice. Pretreatment of the cells with cAMP did not enhance the efflux response to high density lipoprotein isolated from HuA-IVTg mouse serum. Our results suggest that apoA-IV, unassociated with high density lipoprotein particles, is responsible for enhanced cholesterol efflux. This study illustrates the role of lipid-free apolipoproteins in mediating cellular cholesterol efflux with use of a biological fluid and is potentially of physiological relevance, especially in apolipoprotein-rich extravascular fluids.

Paraoxonase activity is reduced by a pro-atherosclerotic diet in rabbits.

Serum paraoxonase (PON1) is believed to protect against the development of atherosclerosis because of its ability to retard the oxidation of low-density lipoprotein (LDL) by hydrolysing LDL-associated phospholipid and cholesteryl-ester hydroperoxides. We have examined the relationship between PON1 and atherosclerosis development in transgenic rabbits overexpressing human apolipoprotein (apo) A-I and nontransgenic littermates fed a pro-atherogenic diet. PON1 activity was higher in transgenic (4006.1 +/- 716.7 nmol/min/ml) compared to control (3078.5 +/- 623.3 nmol/min/ml) rabbits (P < 0.01) while high-density lipoprotein (HDL) cholesterol was 1.84 +/- 0.54 mmol/L in transgenic rabbits and 0.57 +/- 0.21 mmol/L in control rabbits (P = 0.0001). After feeding rabbits a high-cholesterol diet for 14 weeks HDL-cholesterol fell by 70% in both transgenic and control rabbits (P < 0.001 compared to week 0) PON1 activity fell by 50% in both groups of rabbits (P < 0. 01 compared to week 0). The amount of thoracic aortic surface area covered by lesions was 29 +/- 16% in the control group and 26 +/- 15% in the transgenic group (P = NS). A pro-atherosclerotic diet reduces PON1 which may exaggerate the effects of the diet on the development of atherosclerosis.

Complete atherosclerosis regression after human ApoE gene transfer in ApoE-deficient/nude mice.

The apolipoprotein E (apoE)-deficient mouse is a relevant animal model of human atherosclerosis. Although the prevention of atherosclerosis development has been documented after somatic gene transfer into animal models, regression of lesions remains to be demonstrated. Thus, we used this genetically defined mouse model nn the nude background to show atherosclerosis regression. ApoE-deficient nude mice were infected with 5 x 10(8) or 10(9) plaque-forming units of a first-generation adenovirus encoding human apoE cDNA. The secretion of human apoE resulted in a rapid decrease of total cholesterol, which normalized the hypercholesterolemic phenotype within 14 days (from 600+/-100 to <100 microg/mL). Transgene expression was observed during a period of >4 months, with a normalization of cholesterol and triglyceride levels during 5 months. At that time, we successfully reinjected the recombinant adenovirus and observed the appearance of the human protein as well as the correction of lipoprotein phenotype. In mice killed 6 months-after the first infection, we observed a dose-dependent regression of fatty streak lesions in the aorta. We showed sustained expression of a transgene with a first-generation adenoviral vector and a correction of dyslipoproteinemia phenotype leading to lesion regression. These data demonstrate that somatic gene transfer can induce plaque regression.

Clearance of cationized LDL cholesterol from a muscle depot is not enhanced in human apolipoprotein A-IV transgenic mice.

Human apolipoprotein A-IV (apoA-IV) transgenic mice fed an atherogenic diet were shown previously to develop less atherosclerosis than control mice. The question arose whether the antiatherogenic effect of human apoA-IV is due to enhancement of reverse cholesterol transport despite no increase in plasma high-density lipoprotein (HDL) cholesterol. We studied male and female mice overexpressing human apoA-IV and their wild-type (WT) controls, all of which were fed a chow diet. Plasma total and HDL cholesterol and total phospholipids were not increased in the transgenic mice, and regression analysis showed no correlation between plasma levels of cholesterol or phospholipids and plasma human apoA-IV. To study reverse cholesterol transport in vivo, the disappearance of cholesterol from a depot of [(3)H]cholesterol-labeled cationized low-density lipoprotein injected into the rectus femoris muscle was compared in high expressers of human apoA-IV and WT controls. The loss of radioactivity and the diminution of the exogenous cholesterol mass were determined on days 8 and 12 after injection. No enhanced loss of radioactivity or cholesterol mass was seen in the transgenic mice even at levels of 2500 mg/dL of human apoA-IV. In some instances, there was even slower loss of exogenous cholesterol (radioactivity and mass) in the transgenic mice. Although [(3)H]cholesterol efflux from cultured human skin fibroblasts and mouse peritoneal macrophages was only approximately 30% higher in the presence of sera from high expressers of human apoA-IV, addition of phosphatidylcholine liposomes enhanced the efflux in both groups to the same extent. Another paradoxical finding was that the cholesterol esterification rate in plasma was 34% to 36% lower in human apoA-IV mice than in WT controls. In conclusion, even though apoA-IV was found previously to be atheroprotective under hypercholesterolemic conditions, high plasma levels of human apoA-IV did not enhance cholesterol mobilization in vivo in normocholesterolemic mice.

Protective role of interleukin-10 in atherosclerosis.

The potential role of anti-inflammatory cytokines in the modulation of the atherosclerotic process remains unknown. Interleukin (IL)-10 has potent deactivating properties in macrophages and T cells and modulates many cellular processes that may interfere with the development and stability of the atherosclerotic plaque. IL-10 is expressed in human atherosclerosis and is associated with decreased signs of inflammation. In the present study, we show that IL-10-deficient C57BL/6J mice fed an atherogenic diet and raised under specific pathogen-free conditions exhibit a significant 3-fold increase in lipid accumulation compared with wild-type mice. Interestingly, the susceptibility of IL-10-deficient mice to atherosclerosis was exceedingly high (30-fold increase) when the mice were housed under conventional conditions. Atherosclerotic lesions of IL-10-deficient mice showed increased T-cell infiltration, abundant interferon-gamma expression, and decreased collagen content. In vivo, transfer of murine IL-10 achieved 60% reduction in lesion size. These results underscore the critical roles of IL-10 in both atherosclerotic lesion formation and stability. Moreover, IL-10 appears to be crucial as a protective factor against the effect of environmental pathogens on atherosclerosis.

Human ATP-binding cassette transporter 1 (ABC1): genomic organization and identification of the genetic defect in the original Tangier disease kindred.

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.

Apolipoprotein A-I (R151C)Paris is defective in activation of lecithin: cholesterol acyltransferase but not in initial lipid binding, formation of reconstituted lipoproteins, or promotion of cholesterol efflux.

ApoA-I(R151)Paris is a natural apolipoprotein (apo) A-I variant that is associated with low levels of high-density lipoprotein cholesterol (HDL-cholesterol) and the partial deficiency of lecithin:cholesterol acyl-transferase (LCAT) in the plasma of heterozygous carriers. We compared the abilities of recombinant normal apoA-I and recombinant apoA-I(R151C)Paris to clear an emulsion of dimyristoylphosphatidylcholine (DMPC), to form reconstituted lipoproteins with dipalmitoylphosphatidylcholine (DPPC), to activate LCAT, and to promote efflux of biosynthetic cholesterol from porcine aortic smooth muscle cells (SMCs) or of exogenous cholesterol from lipid-loaded mouse peritoneal macrophages. Recombinant apoA-I(R151C)Paris occurred in monomeric and dimeric forms at a ratio of 60:40. Normal apoA-I and apoA-I(R151C)Paris cleared DMPC emulsions at equal rates. Both isoforms associated completely with DPPC during cholate dialysis. Normal apoA-I formed one single particle with a mean diameter of 9.3 nm, whereas apoA-I(R151)Paris gave rise to three particles with mean diameters of 9.3 nm (containing 74% of apoA-I), 10.6 nm, and 12.1 nm, respectively. Compared to normal apoA-I, apoA-I(R151C)Paris had a reduced LCAT-cofactor activity with a 60% lower Vmax/Km ratio due to a 50% higher affinity constant, Km. During incubations for 10 min and 360 min, normal apoA-I/DPPC complexes and apoA-I(R151C)Paris/DPPC complexes were equally efficient in releasing biosynthetic cholesterol from SMCs. In the lipid-free form, apoA-I(R151C)Paris induced normal hydrolysis of cholesteryl esters and normal cholesterol efflux from lipid-loaded mouse-peritoneal macrophages. In conclusion, in addition to its ability to form homo- and heterodimers, apoA-I(R151C)Paris is characterized by defective LCAT-cofactor activity but by normal lipid binding and cholesterol-efflux-promoting abilities.