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The chemical composition and metabolism of lipoproteins in a population of Benedictine nuns were studied after 5-month periods during which the predominant dietary fats were sunflower oil, peanut oil, palm oil, or milk fats (butter and cream). The population was divided into three groups. The control group (C) included twelve subjects selected at random by taking two subjects per age pool among those with plasma cholesterol less than 230 mg/dl. Groups H1 and H2 were selected at random in the same way, among those with plasma cholesterol greater than 230 mg/dl. Each group comprised six subjects and differed from each other in the amount of plasma cholesteryl esters, i.e., below and above the mean value of group C, for H1 and H2, respectively. Changes in the degree of saturation of the predominant fat of the diet were associated with changes in both the chemical composition of lipoproteins and their cellular metabolism studied in fibroblast cultures. No significant difference between the normocholesterolemic subjects of group C and the ''high risk'' subjects of groups H1 and H2 was found.

Scavenger receptor SR-BI has been implicated in HDL-dependent atheroprotective mechanisms. We report the generation of an SR-BI conditional knockout mouse model in which SR-BI gene targeting by loxP site insertion produced a hypomorphic allele (hypomSR-BI). Attenuated SR-BI expression in hypomSR-BI mice resulted in 2-fold elevation in plasma total cholesterol (TC) levels. Cre-mediated SR-BI gene inactivation of the hypomorphic SR-BI allele in hepatocytes (hypomSR-BI-KO(liver)) was associated with high plasma TC concentrations, increased plasma free cholesterol/TC (FC/TC) ratio, and a lipoprotein-cholesterol profile typical of SR-BI-/- mice. Plasma TC levels were increased 2-fold in hypomSR-BI and control mice fed an atherogenic diet, whereas hypomSR-BI-KO(liver) and SR-BI-/- mice developed severe hypercholesterolemia due to accumulation of FC-rich, VLDL-sized particles. Atherosclerosis in hypomSR-BI mice was enhanced (2.5-fold) compared with that in controls, but to a much lower degree than in hypomSR-BI-KO(liver) (32-fold) and SR-BI-/- (48-fold) mice. The latter models did not differ in either plasma lipid levels or in the capacity of VLDL-sized lipoproteins to induce macrophage cholesterol loading. However, reduced atherosclerosis in hypomSR-BI-KO(liver) mice was associated with decreased lesional macrophage content as compared with that in SR-BI-/- mice. These data imply that, in addition to its major atheroprotective role in liver, SR-BI may exert an antiatherogenic role in extrahepatic tissues.

Objective: The human scavenger receptor class B type I (Cla-1) plays a key role in cellular cholesterol movement in facilitating transport of cholesterol between cells and lipoproteins. Indirect evidence has suggested that Cla-1 gene expression is under the feedback control of cellular cholesterol content. To define the molecular mechanisms underlying such putative regulation, we evaluated whether Cla-1 is a target gene of the sterol regulatory element binding protein (SREBP) transcription factor family., Methods and Results: Transient transfections demonstrated that SREBP factors induce Cla-1 promoter activity and that SREBP-2 is a more potent inducer than the SREBP-1a isoform. The 5'-deletion analysis of 3 kb of the 5'-flanking sequence of the Cla-1 gene, combined with site-directed mutagenesis and electrophoretic mobility shift assay, allowed identification of a unique sterol responsive element. SREBP-mediated Cla-1 regulation was confirmed in stably transfected human embryonic kidney 293 cells expressing the active form of SREBP-2 at incremental levels. In these cell lines, Cla-1 mRNA and protein levels were increased in direct proportion to the level of SREBP-2 expression., Conclusions: These findings provide evidence that SREBP-2, a key regulator of cellular cholesterol uptake through modulation of the expression of the low-density lipoprotein receptor gene, may influence cellular cholesterol homeostasis via regulation of Cla-1 gene expression.

Lp(a) concentrations vary considerably among individuals and are primarily determined by the apo(a) gene locus. We have previously shown that mean plasma Lp(a) levels in the chimpanzee are significantly higher than those observed in humans (Doucet, C., Huby, T., Chapman, J., and Thillet, J. (1994) J. Lipid Res 35, 263-270). To evaluate the possibility that this difference may result from a high level of expression of chimpanzee apo(a), we cloned and sequenced 1.4 kilobase (kb) of the 5'-flanking region of the gene and compared promoter activity to that of its human counterpart. Sequence analysis revealed 98% homology between chimpanzee and human apo(a) 5' sequences; among the differences observed, two involved polymorphic sites associated with Lp(a) levels in humans. The TTTTA repeat located 1.3 kb 5' of the apo(a) gene, present in a variable number of copies (n = 5-12) in humans, is uniquely present as four copies in the chimpanzee sequence. The second position concerns the +93 C>T polymorphism that creates an additional ATG start codon in the human apo(a) gene, thereby impairing translation efficiency. In chimpanzee, this position did not appear polymorphic, and a base difference at position +94 precluded the presence of an additional ATG. In transient transfection assays, the chimpanzee apo(a) promoter exhibited a 5-fold elevation in transcriptional activity as compared with its human counterpart. This marked difference in activity was maintained with either 1.4 kb of 5' sequence or the minimal promoter region -98 to +141 of the human and chimpanzee apo(a) genes. Using point mutational analyses, nucleotides present at positions -3, -2, and +8 (relative to the start site of transcription) were found to be essential for the high transcription efficiency of the chimpanzee apo(a) promoter. High transcriptional activity of the chimpanzee apo(a) gene may therefore represent a key factor in the elevated plasma Lp(a) levels characteristic of this non-human primate.

Autosomal dominant type IIa hypercholesterolaemia (ADH) is characterised by an elevation of total plasma cholesterol associated with increased LDL particles. Numerous different molecular defects have been identified in the LDL receptor (LDLR) and few specific mutations in the apolipoprotein B (APOB) gene resulting in familial hypercholesterolaemia and familial defective apoB-100 respectively. To estimate the respective contribution of LDLR, APOB and other gene defects in this disease, we studied 33 well characterised French families diagnosed over at least three generations with ADH through the candidate gene approach. An estimation of the proportions performed with the HOMOG3R program showed that an LDLR gene defect was involved in approximately 50% of the families (P = 0.001). On the other hand, the estimated contribution of an APOB gene defect was only 15%. This low estimation of ADH due to an APOB gene defect is further strengthened by the existence of only two probands carrying the APOB (R3500Q) mutation in the sample. More importantly and surprisingly, 35% of the families in the sample were estimated to be linked to neither LDLR nor APOB genes. These data were confirmed by the exclusion of both genes through direct haplotyping in three families. Our results demonstrate that the relative contributions of LDLR and APOB gene defects to the disease are very different. Furthermore, our results also show that genetic heterogeneity is, generally, underestimated in ADH, and that at least three major groups of defects are involved. At this point, the contribution of the recently mapped FH3 gene to ADH cannot be assessed nor its importance in the group of 'non LDLR/non APOB' families.

A new polymorphism located at position -629 (CETP/-629A/C) in the promoter of the cholesteryl ester transfer protein (CETP) gene is described. The -629A allele was associated with lower CETP mass (P<0. 0001) and higher high density lipoprotein cholesterol (P<0.001) than the C allele in a sample of 536 control subjects from the ECTIM study. Transfection studies in HepG2 cells with a luciferase expression vector incorporating a 777-bp fragment of the CETP promoter and containing either A or C at position -629 showed significantly lower luciferase activity with the promoter fragment of the A allele (-25%, P<0.05). By gel-shift assay, DNA-protein interactions were evaluated in nuclear extracts of HepG2 cells with the use of 2 probes (A or C probe) composed of 20 bp of the promoter sequence surrounding the polymorphic site. Two specific complexes of distinct migration rate were identified with the A and the C probe. Competition with an excess of oligonucleotide containing the Sp1 consensus binding site showed that a protein(s) of the Sp transcription factor family was implicated in complex formation with the A probe but not with the C probe. Incubation with specific antibodies indicated that Sp1 and Sp3 bound specifically to the A probe. We introduced mutations in the -629-Sp1 binding site to test its functionality and to define the characteristics of transcription factor binding. We showed, by gel-shift assay, that no nuclear proteins bound to the mutated sequence. Transient transfection of HepG2 cells revealed that the expression of the mutated fragment was significantly increased compared with that of the A promoter fragment (25%, P<0.05). The mutated fragment displayed the same activity as that of the C promoter. These results indicate that Sp1 and/or Sp3 repress CETP promoter activity, whereas nuclear factors binding the C allele are without effect on promoter expression.

Background: To define the role of metalloproteinases (MMPs) in the development of lipid-rich atherosclerotic lesions in relation to the balance between proteolytic and antiproteolytic activities, we investigated the impact of adenovirus-mediated elevation in the circulating levels of human tissue inhibitor of MMP (TIMP-1) in atherosclerosis-susceptible apolipoprotein E-deficient (apoE(-/-)) mice., Methods and Results: Infusion of apoE(-/-) mice fed a lipid-rich diet with rAd.RSV.TIMP-1 (1x10(11) viral particles) resulted in high hepatic expression of TIMP-1. At 2 weeks after injection, plasma TIMP-1 levels ranged from 7 to 24 micrograms/mL (mean 14.8+/-6.8). Marked overexpression of TIMP-1 was transient, with levels of TIMP-1 decreasing to 2.5 to 8 micrograms/mL (mean 4.3+/-2.1) at 4 weeks. Plasma lipid and lipoprotein levels in mice treated with rAd.RSV.TIMP-1 were similar to those treated with rAd.RSV.betaGal. However, rAd.RSV.TIMP-1-infused mice displayed a marked reduction (approximately 32%; P<0.05) in mean lesion area per section (512+/-121 micrometers(2)x10(3); n=12 sections from 4 animals) as compared with rAd.RSV.betaGal-infused mice (750+/-182 micrometers(2)x10(3); n=12 sections from 4 animals). Similarly, marked reduction in macrophage deposition as well as MMP-2, MMP-3, and MMP-13 antigens was observed., Conclusions: Histological and immunohistologic analyses of atherosclerotic lesions revealed increases in collagen, elastin, and smooth muscle alpha-actin content in mice treated with rAd.RSV.TIMP-1. These qualitative and quantitative features were the consequence of TIMP-1 infiltration from plasma to arterial intima, as immunohistochemical analyses revealed an abundance of TIMP-1 specifically in lesions of rAd.RSV. TIMP-1-treated mice.

Lipid-laden macrophages express tissue factor (TF), which may activate the extrinsic coagulation pathway on rupture of the atherosclerotic plaque. Tissue factor pathway inhibitor (TFPI) is a major regulator of TF-induced coagulation. We evaluated the possibility that monocyte-derived macrophages express this protein, thereby contributing to regulation of TF activity (TFact). Equally, we investigated the effect of cholesterol and of oxidized LDL (Ox-LDL) on the expression of TFPI and TF by human monocyte-derived macrophages (HMDMs). Northern blot analysis of TFPI mRNA from cultured HMDMs revealed a single band at 4.2 kb with weak intensity; this finding was confirmed by reverse transcription-polymerase chain reaction. Gel filtration of HMDM supernatants showed the presence of an active 100-kDa form of TFPI, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions; under reducing conditions, however, the immunoblot revealed a 40-kDa form of TFPI. The TFPI in HMDM supernatants possessed heparin-binding affinity, suggesting potential interaction of TFPI with heparan sulfate proteoglycans. Stimulation of foam cell formation by incubation of macrophages for 48 hours with exogenous free cholesterol indicated that neither the biological activity nor the de novo synthesis of TFPI protein was affected. In contrast, cholesterol loading with exogenous free cholesterol induced significant upregulation of total TFact (2.6-fold: 25.0 versus 9.4 mU/mg cell protein, cholesterol-treated versus control cells; P<0. 05); such induction was not correlated with an elevation in TF antigen (8.5 versus 7.8 ng/mg cell protein, cholesterol-treated versus control cells). Similarly, cholesterol-rich Ox-LDL induced an increase in TFact (1.9-fold: 18.9 versus 10.0 mU/mg cell protein, Ox-LDL-treated versus control cells; P<0.05); by contrast, the amount of TF antigen remained unchanged (7.1 versus 7.9 ng/mg cell protein, Ox-LDL-treated versus control cells). Our data indicate that enhancement of the procoagulant activity of TF in macrophage-derived foam cells is not counterbalanced by upregulation of TFPI activity, suggesting that lesion foam cells are in a procoagulant state; they may therefore contribute to thrombus generation on plaque rupture.

The regulation of macrophage lipoprotein lipase (LPL) secretion and mRNA expression by atherogenic lipoproteins is of critical relevance to foam cell formation. LPL is present in arterial lesions and constitutes a bridging ligand between lipoproteins, proteoglycans, and cell receptors, thus favoring macrophage lipoprotein uptake and lipid accumulation. We investigated the effects of native and of oxidized lipoproteins on the expression of LPL in an in vitro human monocyte-macrophage system. Exposure of mature macrophages (day 12) to highly copper-oxidized human low density lipoprotein (LDL) (100 microg protein per milliliter) led to marked reduction in the expression of LPL activity (-62%, P<0.01) and mRNA level (-47%, P<0.05); native LDL, acetylated LDL, and LDL oxidized for <6 hours were without effect. The reduction in LPL activity became significant at a threshold of 6 hours of LDL oxidation (-31%, P<0.05). Among the biologically active sterols formed during LDL oxidation, only 7beta-hydroxycholesterol (5 microg/mL) induced a minor reduction in macrophage LPL activity, whereas 25-hydroxycholesterol was without effect. By contrast, lysophosphatidylcholine, whose LDL content increased in parallel with the degree of oxidation, induced significant reductions in LPL activity and mRNA levels at concentrations of 2 to 20 micromol/L (-34% to -53%, P<0.01). Our results demonstrate that highly oxidized LDL (>6-hour oxidation) exerts negative feedback on LPL secretion in human monocytes-macrophages via a reduction in mRNA levels. By contrast, native LDL and mildly oxidized LDL (<6-hour oxidation) did not exert a feedback effect on LPL expression. We speculate that the content of lysophosphatidylcholine and, to a lesser degree, of 7beta-hydroxycholesterol in oxidized LDLs is responsible for the downregulation of LPL activity and mRNA abundance in human monocyte-derived macrophages and may therefore modulate LPL-mediated pathways of lipoprotein uptake during conversion of macrophages to foam cells.

Calcium antagonists and beta-blockers may retard or inhibit atherogenesis. In the absence of data pertaining to the potential cardioprotective action of an association of such agents, we have investigated the impact of nifedipine and atenolol, alone or in combination, on the capacity of monocyte-macrophages (ex vivo) and copper ions (in vitro) to oxidize LDL and on intracellular metabolism and efflux of free and esterified forms of cholesterol in human macrophages and foam cells. At concentrations up to 100 micromol/L, atenolol had no effect on the oxidative resistance of LDL; on the contrary, nifedipine displayed a significant dose-dependent capacity to protect LDL during copper-mediated oxidation (100 micromol/L; P<.001). Using a DPPH radical generating system, nifedipine was shown to exert free radical-trapping activity (molar ratio of scavenging activity, nifedipine:alpha-tocopherol, 1:114). The addition of atenolol to nifedipine was without effect on the antioxidant activity of the calcium antagonist. In experiments in which oxidative modification was mediated by monocyte-macrophages, nifedipine but not atenolol conserved its antioxidant capacity. Furthermore, we demonstrated that association of atenolol with nifedipine did not modify the antioxidant properties of nifedipine itself. Using a human monocyte-derived macrophage culture system, nifedipine, atenolol, or a combination of the two drugs was ineffective in inhibiting foam cell formation induced by acetylated LDL or oxidized LDL. However, atenolol (100 micromol/L) increased cellular accumulation of cholesteryl ester (+17%; P<.05), whereas nifedipine (100 micromol/L) decreased total cholesterol (-37.4%; P<.05) accumulation induced by acetylated LDL in the mouse macrophage cell line J774. A combination of the two drugs neutralized these antagonistic effects. None of these results were reproduced during the oxidized LDL-induced transformation of murine J774 cells into foam cells. Furthermore, cholesterol efflux from preloaded human macrophages was equally unaffected by the addition of the drugs alone or in combination. It therefore seems unlikely that the beneficial effect of atenolol on coronary heart disease is mediated by changes in either LDL oxidizability or cholesterol metabolism in human macrophages and foam cells. Our findings with nifedipine suggest, however, that this calcium antagonist may potentially exert antiatherosclerotic properties via a reduction of the oxidative modification of LDL, thereby affecting a reduction in foam cell formation and in the pathophysiological cellular activities of oxidized lipids, rather than by inducing a direct reduction in cholesterol accumulation in human foam cells of macrophage origin.

The presence of lecithin:cholesterol acyltransferase (LCAT) deficiency in six probands from five families originating from four different countries was confirmed by the absence or near absence of LCAT activity. Also, other invariate symptoms of LCAT deficiency, a significant increase of unesterified cholesterol in plasma lipoproteins and the reduction of plasma HDL-cholesterol to levels below one-tenth of normal, were present in all probands. In the probands from two families, no mass was detectable, while in others reduced amounts of LCAT mass indicated the presence of a functionally inactive protein. Sequence analysis identified homozygous missense or nonsense mutations in four probands. Two probands from one family both were found to be compound heterozygotes for a missense mutation and for a single base insertion causing a reading frame-shift. Subsequent family analyses were carried out using mutagenic primers for carrier identification. LCAT activity and LCAT mass in 23 genotypic heterozygotes were approximately half normal and clearly distinct from those of 20 unaffected family members. In the homozygous patients no obvious relationship between residual LCAT activity and the clinical phenotype was seen. The observation that the molecular defects in LCAT deficiency are dispersed in different regions of the enzyme suggests the existence of several functionally important structural domains in this enzyme.

The chemical composition and metabolism of lipoproteins in a population of Benedictine nuns were studied after 5-month periods during which the predominant dietary fats were sunflower oil, peanut oil, palm oil, or milk fats (butter and cream). The population was divided into three groups. The control group (C) included twelve subjects selected at random by taking two subjects per age pool among those with plasma cholesterol less than 230 mg/dl. Groups H1 and H2 were selected at random in the same way, among those with plasma cholesterol greater than 230 mg/dl. Each group comprised six subjects and differed from each other in the amount of plasma cholesteryl esters, i.e., below and above the mean value of group C, for H1 and H2, respectively. Changes in the degree of saturation of the predominant fat of the diet were associated with changes in both the chemical composition of lipoproteins and their cellular metabolism studied in fibroblast cultures. No significant difference between the normocholesterolemic subjects of group C and the "high risk" subjects of groups H1 and H2 was found.

Human low density lipoproteins are metabolized by cultured human fibroblasts, through a specific metabolic pathway, which entails the regulation of intracellular cholesterol synthesis. When anti-lipoprotein IgA coming from patients with myeloma, mixed hyperlipidaemia and xanthomatosis were introduced into the system, we observed a decrease of the protein degradation of the LDL molecule, and a disappearance of the regulation of intracellular cholesterol synthesis. In the same system, an anti-lipoprotein IgA from a case of myeloma with mixed hyperlipidaemia, but without xanthomatosis, or control IgG and IgA were inactive and did not modify the LDL pathway.