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In addition to its effects on cholesterol levels, apoE3 has lipid-independent effects that contribute to cardiovascular protection; one of these effects is the ability to inhibit cell cycling in VSMCs. The goal of this study was to identify and characterize cell cycle-regulatory mechanisms responsible for the anti-mitogenic effect of apoE.Primary VSMCs were stimulated with serum in the absence or presence of apoE3. apoE3 upregulated expression of the cdk inhibitor, p27(kip1), in primary VSMCs, and this effect required Cox2 and activation of PGI(2)-IP signaling. The microRNA family, miR221/222 has recently been identified as a post-translational regulator of p27, and apoE3 inhibited miR221/222 expression in a Cox2- and PGI(2)/IP-dependent manner. Moreover, reconstituted miR222 expression was sufficient to override the effects of apoE on p27 expression and S phase entry. The ability to repress expression of miR221/222 is shared by apoE3-containing HDL but is absent from apoA-1, LDL and apoE-depleted HDL. All three apoE isoforms regulate miR221/222, and the effect is independent of the C-terminal lipid-binding domain. miR221/222 levels are increased in the aortae of apoE3-null mice and reduced when apoE3 expression is reconstituted by adeno-associated virus infection. Thus, regulation of miR221/222 by apoE3 occurs in vivo as well as in vitro.ApoE inhibits VSMC proliferation by regulating p27 through miR221/222. Control of cell cycle-regulatory microRNAs adds a new dimension to the spectrum of cardiovascular protective effects afforded by apoE and apoE-HDL.

Objectives The aim of the present study was to determine hepatic paraoxonase 1 (PON1) status in response to apoE genotype and dietary quercetin supplementation in mice. Methods and results ApoE3 and apoE4 transgenic mice were fed semi-synthetic diets without (controls) and with quercetin (2 mg/g diet) for 6 weeks. Hepatic mRNA and protein levels of PON1 were significantly lower in apoE4 as compared to apoE3 mice. Feeding quercetin-enriched diets induced hepatic PON1 gene expression with a tendency for greater induction in apoE3 as compared to apoE4 mice. Furthermore, hepatic mRNA and protein levels of β-glucuronidase and sulfatase, both enzymes centrally involved in the deconjugation of quercetin conjugates, were lower in apoE4 vs. apoE3 mice. PPARγ (which partly controls PON1 gene expression) mRNA levels were lower in apoE4 vs. apoE3 mice. Conclusion We provide first evidence that PON1 is differentially regulated in response to apoE genotype.

Transglutaminases play an important role in vascular remodeling, calcification, cell adhesion and endothelial barrier function. In this study we investigate the influence of combined inhibition of both tissue-type transglutaminase (TG2) and the plasma transglutaminase FXIIIA on early lesion development. Methods: A cuff was placed around the femoral arteries of ApoE3 Leiden mice while fed a Western type diet to induce atherosclerotic lesion development. An osmotic minipump was placed in the intraperitoneal cavity containing an irreversible inhibitor of TG2 and FXIIIA activity ((1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride, Zedira). Atherosclerotic lesion composition was analyzed using immunohistochemistry and RT-PCR. Results: Inhibition of transglutaminases did not influence lesion size or geometric remodeling of the vessels. However, systemic transglutaminase inhibition resulted in 41% less macrophage infiltrate in the media of the vessels. Additional in vitro experiments on HL60 cells confirmed a decreased migratory response during transglutaminase inhibition. Conclusion: Inhibition of TG2 and FXIIIA during early development of lesions reduced the macrophage content in the media of atherosclerotic vessels, while not affecting lesion size or geometric remodeling. (C) 2010 Elsevier Ireland Ltd. All rights reserved

Inflammation is crucial in all stages of atherosclerosis, and few studies have investigated the effect of dietary fat on markers of inflammation related to this disease during the postprandial period.To evaluate the chronic effects of dietary fat on the postprandial expression of proinflammatory genes in peripheral blood mononuclear cells (PBMCs) in healthy subjects.20 healthy men followed three different diets for 4 weeks each, according to a randomized crossover design: Western diet: 15% protein, 47% carbohydrates (CHO), 38% fat (22% saturated fatty acid (SFA)); Mediterranean diet: 15% protein, 47% CHO, 38% fat (24% monounsaturated fatty acid (MUFA)); CHO-rich and n-3 diet: 15% protein, 55% CHO,30% fat (8% polyunsaturated fatty acid (PUFA)). After 12-h fast, volunteers were given a breakfast with a fat composition similar to that consumed in each of the diets-butter breakfast: 35% SFA; olive oil breakfast: 36% MUFA; walnut breakfast: 16% PUFA, 4% alpha-linolenic acid (LNA).The butter breakfast induced a higher increase in tumor necrosis factor (TNF)-alpha messenger RNA (mRNA) expression than the olive oil or walnut breakfasts (P=0.014) in PBMCs. Moreover, we found a higher postprandial response in the mRNA of interleukin (IL)-6 with the intake of butter and olive oil breakfasts than with the walnut breakfast (P=0.025) in these cells. However, the effects of the three fatty breakfasts on the plasma concentrations of these proinflammatory parameters showed no significant differences (P=N.S.).Consumption of a butter-enriched meal elicits greater postprandial expression of proinflammatory cytokine mRNA in PBMCs, compared to the olive oil and walnut breakfasts.

Hepatic gene transfer of atheroprotective human apoE by recombinant viral vectors can reverse hypercholesterolaemia and inhibit atherogenesis in apoE-deficient (apoE(-/-)) mice. Here, in preliminary studies we assess the effectiveness of a recently developed self-complementary adeno-associated virus (scAAV) serotype 8 vector, driven by a hepatocyte-specific promoter (LP1), for liver-directed gene delivery of human apoE3. Vector viability was validated by transducing cultured HepG2 cells and measuring secretion of apoE3 protein. Male and female apoE(-/-) mice, 6-month old and fed on normal chow, were intravenously injected with 1x10(11) vg (vector genomes) of scAAV2/8.LP1.apoE3; age-matched untreated mice served as controls. In male mice, plasma apoE3 levels were sufficiently high (up to 17 microg/ml) to normalize plasma total cholesterol and ameliorate their proatherogenic lipoprotein profile, by reducing VLDL/LDL and increasing HDL 5-fold. At termination (12 weeks) development of aortic atherosclerosis was significantly retarded by 58% (aortic lesion area 8.2+/-1.4% vs. 19.3+/-2.4% in control males; P

Apolipoprotein (apoE) E is a multifunctional protein that plays a critical role in atherogenesis, in part by regulating the intimal proliferation of vascular smooth muscle cells. Recently, a novel cyclooxygenase (COX)-2 pathway was shown to contribute to the anti-proliferative action of human apoE3 in vascular smooth muscle cells (VSMC). Here, we provide insight into the structure-function properties by which apoE mediates these effects. ApoE3 is most effective in promoting COX-2 expression as a lipid-free protein and is less active after lipidation. Alterations in the stability of the helix-bundle N-terminal domain of apoE that contains the binding site for the low density lipoprotein (LDL) receptor and heparin do not affect the up-regulation of the COX-2 pathway. In addition, the apoE2, 3, and 4 isoforms are all capable of up-regulating the COX-2 pathway. Finally, the effect of apoE on COX-2 was found to be independent of expression on the VSMC surface of the LDL receptor and heparan sulfate proteoglycans (HSPG). In summary, our data indicates that apoE, particularly in the lipid-free state, can up-regulate COX-2 in murine vascular smooth muscle cells apparently independently of binding to the LDLR, LRP or HSPG.

Apo E plays an important role in chylomicron and VLDL remnant processing, uptake or conversion to LDL. The type of lipoprotein that isolates in the LDL density of E2/2 subjects was investigated and the effect of the apo E isoforms on LDL mass was determined in all genotypes in a large group of Type 1 diabetics. Analysis of the LDL composition of E2/2 homozygotes (n = 6) compared to subjects with the common E3/3 isoform (n = 6) demonstrated an enrichment in apo E, unesterified cholesterol, phospholipid and triglyceride relative to apo B in E2/2 subjects, more typical of a dense IDL remnant than of LDL. Although diabetics were studied, these findings are considered to reflect those of the general population. Comparison of the lipoprotein distribution of homozygous and heterozygous subjects revealed that, as genotype changed from E4/4 (n = 22) to E3/4 (n = 262), E3/3 (n = 710) = E2/4 (n = 30), E2/3 (n = 151), E2/2 (n = 6), LDL cholesterol decreased significantly in a stepwise manner. The decrease was not in a specific subgroup of LDL. In conclusion, for E2/2 subjects, lipoproteins isolated in the LDL density range appear to be composed mainly of dense IDL remnants and some Lp(a). The apo E isoform also has a significant effect on LDL concentration in both homozygotes and heterozygotes.

The allelic variation of Apolipoprotein E (ApoE) influences serum lipid levels. Postmenopausal estrogen replacement therapy (ERT) has favorable effects on the serum lipid profile. We examined the effect of ApoE genotype on lipid response to ERT in 692 community-dwelling women aged 60 and older. ApoE genotypes were categorized into three groups: ApoE 2 (E2/E2+E2/E3, n=94), ApoE 3 (E3/E3, n=430), and ApoE 4 (E3/E4+E4/E4, n=142). Compared to 497 women not using ERT, 169 women currently using ERT were younger (P=0.01), had lower levels of total cholesterol (TC; P=0.10) and low-density lipoprotein (P0.001), higher levels of high-density lipoprotein (HDL; P0.001) and triglycerides (TG; P=0.009), and were more likely to have had a surgical menopause (P0.001). No significant differences in body mass index, alcohol intake, physical activity, or cigarette smoking were found between current ERT users and nonusers (P0.10). There was a significant interaction between ApoE 2 and ERT for HDL levels: women with ApoE 2 using ERT had the highest HDL levels, and women with ApoE 2 not using ERT had the lowest HDL levels (P=0.015). The unfavorable effect of ApoE 4 genotype on lipoproteins is not altered by HRT, but ApoE 2 genotype modulates the HDL-ERT association in older women.

In a previous study it was shown that postprandial lipid metabolism is delayed in individuals with intra-abdominal visceral fat accumulation. Population studies have shown that as compared with individuals with apolipoprotein (apo) E3/3, those with phenotype apo E3/4 phenotype have higher plasma and low density lipoprotein (LDL)-cholesterol (C) concentration and increased susceptibility to coronary heart disease. The aim of the present study is to determine how apo E4 affects postprandial lipid metabolism by comparing individuals with apo E3/4 to those with apo E3/3 phenotype matched for abdominal visceral fat. Sixty-two Japanese subjects (41 male, 21 female) [average age 48+/-14 years; mean body mass index (BMI) 25+/-5.6 kg/m2] were recruited for this study. The subjects were divided into two groups: those with apo E3/3 (n=43) and those with apo E3/4 phenotype (n=19), as determined by isoelectric focusing (IEF). Visceral fat accumulation was analyzed as area of fat deposition by computerized tomography at the umbilicus level. After a 12-h overnight fasting, an oral vitamin A and a fatty meal were administered to these subjects. The plasma triglyceride (TG) increased significantly hours after fat loading in both groups but the levels of TG were significantly higher in apo E3/4 than in apo E3/3 phenotype at 2, 4 and 6 h after fat loading. Plasma retinyl palmitate (RP) levels were also significantly higher in individuals with apo E3/4 than in those with apo E3/3 phenotype at 2, 4 and 6 h after fat loading. This investigation was then conducted in both genders separately, and found that these associations were statistically significant in men. Furthermore, after matching men for fasting TG levels, these associations did not persist for plasma TG levels at any time point, while plasma RP levels were still significantly higher in apo E3/4 group at 2 and 6 h after fat loading. These results indicate that in Japanese population especially for men apo E phenotype E3/4 is associated with an impaired postprandial TG-rich lipoprotein metabolism relative to apo E3/3 phenotype when matched for intra-abdominal visceral fat accumulation, which has a substantial effect on the metabolism of plasma TG-rich lipoproteins.

Apolipoprotein E3-Leiden (APOE3-Leiden) transgenic mice develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. We have studied the progression and regression of atherosclerosis using immunohistochemistry. Female transgenic mice were fed a moderate fat diet to study atherosclerosis over a longer time period. Fatty streaks arose in the intima and consisted of lipid filled macrophages which differed in origin. All macrophages expressed the macrophage scavenger receptor while two thirds expressed sialoadhesin and were positive for an antibody recognizing marginal zone macrophages (MOMA-1). All macrophages were negative for the scavenger receptor MARCO and 50% were positive for CD4. Small fatty streaks contained CD-3 positive T-lymphocytes which were for more than 70% CD4-positive. ICAM- 1 was positive both in atherosclerotic and control mice. In early plaques, fibrosis was observed on the luminal and medial site of the foam cells while smooth muscle cells were only observed in the fibrous cap. To study regression, we used a high fat, high cholesterol diet to rapidly induce atherosclerosis (14 weeks). The animals were then fed normal chow. Subsequently, atherosclerosis was assayed over time (4, 8, 16 weeks). Cholesterol levels dropped in 4 weeks to control levels. The animals did not show a significantly decrease in plaque size over time, but the percentage macrophages was significantly smaller in the animals after 4 weeks. In conclusion, the APOE3-Leiden mouse is a useful model to study the progression and regression of atherosclerosis.

The purpose of this study was to concurrently assess the relationship of Apolipoprotein E (APOE) with both dementias and vascular illnesses in the very old. Nine hundred and fifty nine subjects (mean age 85 years) in a long-term care facility were genotyped and cognitively tested with the Mini Mental State Exam. All subjects were studied for the relationship of APOE with atherosclerotic heart disease, hypertension, or stroke without concomitant dementia. Four hundred fifty individuals met criteria for inclusion into one of the following groups: Alzheimer's disease (n = 318), vascular dementia (n = 49), or not demented controls (n = 83) and were investigated for the relationship between APOE and these diagnostic categories. APOE epsilon4 was not associated with atherosclerotic heart disease, hypertension, or stroke without concomitant dementia. The APOE epsilon3 allele was more common in men with atherosclerotic heart disease. In contrast, the APOE epsilon4 allele was more common in patients with Alzheimer's disease (22%) and vascular dementia (26%) than in not demented controls (7%). APOE epsilon4 is associated with dementias in the very old, whereas its relationship with either peripheral or central nervous system vascular disease without dementia is not as robust.

Lipoprotein lipase (LPL) is bound to heparan sulphate proteoglycans (HSPG) at the luminal surface of endothelium. It is the key enzyme involved in the hydrolysis of very low density lipoproteins (VLDL). Prior to lipolysis by LPL, the lipoproteins are considered to interact with vessel wall HSPG. Apolipoprotein (apo) E is thought to mediate this interaction thereby enhancing the stability of the lipoprotein-LPL complex. We hypothesize that apo E mutations may cause a diminished interaction of VLDL with HSPG leading to impaired lipolysis of VLDL by HSPG-bound LPL. In order to test this hypothesis, lipolysis experiments were performed using HSPG-bound LPL. The mean lipolysis rates of VLDL, isolated from the apo E2 (Lys146 → Gln) heterozygotes, apo E2 (Arg158 → Cys) homozygotes and apo E3-Leiden heterozygotes were 92.3 ± 10.3 (ns), 77.3 ± 4.2 (P < 0.05) and 76.7 ± 10.0% (P < 0.05), respectively, of that of control VLDL (100.0 ± 9.7%). No differences in lipolysis were observed between VLDL from controls and VLDL from the same patients if LPL in solution was used. Thus, compositional differences alone can not explain the differences in lipolysis rates observed with HSPG-bound LPL. In competition experiments, the binding efficiency to HSPG-LPL of VLDL from the apo E2 (Lys146 → Gln) heterozygotes, apo E2 (Arg158 → Cys) homozygotes and apo E3-Leiden heterozygotes was 63 (ns), 41 (P< 0.05) and 35% (P < 0.05), respectively of that of control VLDL (100%). We conclude that VLDL isolated from apo E2 homozygotes and apo E3-Leiden heterozygotes display decreased lipolysis by HSPG-bound LPL due to a defective binding of these lipoproteins to the HSPG-LPL complex.

Apo E3-leiden transgenic mice express human dysfunctional apo E variant and develop hyperlipidemia and atherosclerosis on a high fat/high cholesterol diet. We characterized diet-induced atherosclerotic lesions in apo E3-leiden transgenic mice using immunocytochemical methods in order to examine foam cell formation and determine whether advanced atherosclerotic lesions develop in these animals. Special attention was given to the presence of oxidized lipoproteins and expression of lipoprotein receptors. Plasma cholesterol levels in apo E3-leiden mice on an atherogenic diet increased from 2 to 36 mmol/l in 4 months. At this time apo E3-leiden mice had developed lesions, which ranged from early fatty streaks in thoracic and abdominal aorta to advanced lesions in aortic arch. Early fatty streaks were entirely composed of macrophages which also expressed scavenger receptors. Epitopes characteristic of oxidized LDL were present in macrophage-rich foam cells. Advanced atherosclerotic lesions also developed in apo E3-leiden mice including smooth muscle cell cap formation and erosion of the media. Macrophages and epitopes characteristic of oxidized LDL were present in core and shoulder regions. Scavenger receptors were expressed in macrophages in advanced lesions, whereas LDL-receptor-related protein (LRP) was mainly expressed in smooth muscle cells. It is concluded that: (1) macrophages are the major cell type in both early and advanced atherosclerotic lesions; (2) scavenger receptors and oxidized lipoproteins are present in lesion macrophages; and (3) LRP is mostly expressed in smooth muscle cells. Thus, lesions in apo E3-leiden transgenic mice have features in common with human atherosclerosis. Since lesion macrophages also retain their ability to synthesize endogenous apo E, apo E3-leiden transgenic mouse may be a useful model for studies on the development and genetics of atherosclerosis.

Apolipoprotein (apo) E7 was originally identified by Yamamura et al. in subjects with atherosclerotic cardiovascular diseases (J. Clin. Invest. 1984;74:1229). However, the lipoprotein abnormalities associated with apo E7 phenotype have not been elucidated. In the current study, to clarify the physiological roles of apo E7, lipoprotein abnormalities were studied in 12 apo E7 heterozygotes. A total of seven subjects were hyperlipidemic and five subjects were normolipidemic. The apo E phenotype was apo E7/3 in 11 subjects and apo E7/4 in one subject. Polymerase chain reaction revealed that all of the subjects with apo E7 phenotype had the same mutation as that of apo E Suita as reported previously (J. Biochem. 1989;105:249). All the hyperlipidemic subjects were over 40 years of age and two of them also had and severe coronary heart disease. Ultracentrifugal analysis revealed that the cholesterol level both in very low density lipoprotein and in intermediate density lipoprotein (IDL) was substantially higher in hyperlipidemic apo E7 heterozygotes, compared with control subjects and that the IDL cholesterol was also increased even in normolipidemic apo E7 heterozygotes. Polyacrylamide gel electrophoresis of lipoproteins showed a midband, which implies the increase of remnant lipoproteins, in 11 subjects out of 12, irrespective of the presence or absence of hyperlipoproteinemia. In two cases, a broad β pattern was observed similar to that seen in type III hyperlipoproteinemia. Dietary therapy was dramatically effective for the treatment of hyperlipidemia in patients with apo E7. These findings confirm that apo E is crucial for remnant lipoprotein metabolism and that apo E7 is related to the increase in serum remnant lipoproteins, which leads to hyperlipoproteinemia in association with obesity, aging and impaired glucose metabolism.

The single nucleotide polymorphisms (SNPs) apolipoprotein E (APOE) epsilon3/epsilon2/epsilon4, cholesteryl ester transfer protein (CETP) TaqIB, and apolipoprotein C3 (APOC3) -482 CT have been associated with an atherogenic lipid profile and, in some studies, with increased cardiovascular risk. However, no data exist on their combined impact on atherosclerotic disease. We therefore aimed at investigating the combined impact of these SNPs on the presence of angiographically determined coronary artery disease (CAD). Genotyping was performed in 557 consecutive Caucasian patients undergoing coronary angiography for the evaluation of CAD. From the individual SNPs, only the APOE epsilon3epsilon4/epsilon4epsilon4 genotype was significantly associated with an increased risk of significant coronary stenoses with lumen narrowingor=50% (odds ratio (OR)=1.77 [1.16-2.71]; p=0.008). However, the risk of CAD strongly increased when more than one of the analysed genetic variants was present: ORs were 2.74 [1.29-5.83]; p=0.009 for patients with both the APOE epsilon3epsilon4/epsilon4epsilon4 and the CETP B1B1 genotype, 1.97 [1.06-3.66]; p=0.031 for patients with both the APOE epsilon3epsilon4/epsilon4epsilon4 genotype and the APOC3 -482T allele, 2.12 [1.31-3.44]; p=0.002 for patients with both the CETP B1B1 genotype and the APOC3 -482T allele, and 3.99 [1.57-13.79]; p=0.029 for patients with all three variants. Multivariate analyses confirmed these results. We conclude that there are strong synergistic effects of the APOE epsilon3/epsilon2/epsilon4, the CETP TaqIB, and the APOC3 -482 CT polymorphisms on their association with CAD.

The mechanisms of diet induced hyperlipidemia and atherosclerosis have been widely studied by delineating the role of candidate genes in transgenic and gene targeted mouse models. However, diet induced hyperlipidemia represents a complex process determined by many lipid genes that is only partly understood. This study is aimed at delineating the events induced by dietary intervention in different mouse models at the level of gene expression using microarray analysis. The focus is on the liver as the organ primarily responding to diet, and crucial in determining plasma lipid levels. Firstly, the effect of the genotype was studied. Expression profiles of liver genes were compared between APOE3Leiden (E3L), APOE knockout (E-/-) and C57BL/6JIco (B6) mice using the Incyte GEM 2.03 array carrying 9552 genes. Several hundred differentially expressed genes were identified indicating that the genotype alone effects gene expression. Secondly, the response of E3L mice to high-fat feeding was investigated using a mild and severe high-fat diet (diet W and N, respectively). Diet W caused differential regulation of 200 genes, while diet N affected the expression of 788 genes in B6 and 1010 genes in E3L mice. Annotation of these genes using the Gene Ontology (GO) database showed that two major processes were strongly affected by genotype and diet, namely lipid metabolism and inflammation, the latter as determined by "immune/defense response and detoxification" processes. Many nuclear receptor target genes were differentially regulated, with the largest effects modulated by the severe high-fat diet N, leading to the suppression of genes involved in bile acid, sterol, steroid, fatty acid, and detoxification metabolism. Strikingly, a substantial part of these nuclear receptor target genes were commonly regulated during the different experimental conditions. The common regulation of many nuclear receptor target genes underlying lipid and detoxification processes as found in this study, suggest a defense mechanism involving many nuclear receptors to protect against the accumulation of toxic endogenous lipids and bile acids. These results further strengthen the close link between hyperlipidemia and inflammatory processes.

Numerous animal studies have reported that garlic can protect against atherosclerosis. However, a comparable number of studies do not support this observation. This contradiction may result from differences in study design, use of different animal models, and use of different garlic formulations and preparations. Here, we investigated the effect of the chemically well-characterized and production-controlled garlic powder printanor on atherosclerosis in the APOE*3-Leiden transgenic mouse, a mouse model well suited for evaluating anti-atherosclerotic properties of drugs and food components under human-like conditions. APOE*3-Leiden mice were fed a Western diet supplemented with either 5 or 50 g kg −1 printanor. As a reference, the commercially available fermented garlic kyolic was included (1.6 g kg −1 diet). Treatment with printanor demonstrated reduced body weight, coinciding with increased feces production and fecal fatty acids excretion. Printanor and kyolic treatment did not affect plasma lipids, markers of inflammation (serum amyloid A, serum-soluble intercellular adhesion molecule-1, and blood-leukocytes tumor necrosis factor- (TNF) production) and vascular activation (plasma von Willebrand factor (vWF)). As analyzed after 28 weeks of treatment, printanor and kyolic did not affect atherosclerotic lesion type, area or composition. Under conditions relevant to the human situation, the well-characterized and production-controlled garlic powder printanor does not display hypolipidemic, anti-inflammatory or anti-atherosclerotic properties. © 2004 Elsevier Ireland Ltd. All rights reserved.

We have investigated the association between APOE genotypes and C-reactive protein (CRP) levels in a cohort of approximately 600 individuals who were candidates for statin therapy. An association had been previously reported between the APOE3 allele and elevated CRP levels. That study only examined men. We have reproduced that association in men and have extended the finding to women. We also investigated the effect of the interaction between APOE genotype and hormone replacement therapy (HRT) status on CRP levels, adjusting for body mass index (BMI) and other covariates. BMI and HRT are also significant predictors of CRP, as previously reported. The effect of HRT is strong enough that the contribution of APOE genotype is no longer statistically significant among women on HRT. We also demonstrate that the presence or absence of the single SNP Cysl30Arg (which distinguishes APOE4 from APOE2 and APOE3) is sufficient to determine whether an individual is predisposed to higher or lower CRP levels. Essentially, the presence of one or two copies of APOE4 is associated with a reduction of CRP levels by approximately 34% relative to individuals with zero copies (1.73 mg/L for subjects with one or two copies versus 2.63 mg/L for subjects with zero copies of APOE4). We also tested previously reported associations between CRP levels and polymorphisms in the CRP and IL6 genes. These associations were not reproduced in our cohort.

Raman spectroscopy allows quantitative, non-destructive evaluation of entire, intact atherosclerotic plaques. We quantified the anti-atherosclerotic effects of atorvastatin and amlodipine on progression of atherosclerosis using post-mortem Raman spectroscopic plaque imaging in 28 APOE*3 Leiden transgenic mice who were fed a high fat/high cholesterol diet for 28 weeks. Mice were assigned to a control group receiving the diet alone or to groups that received the diet with either 0.01% w/w atorvastatin, 0.002% w/w amlodipine, or the combination. The entire excised aortic arch was scanned with Raman microspectroscopy for quantitation of the distribution of cholesterol and calcification content. When mice had been treated with atorvastatin, cholesterol accumulation and calcification in the aortic arch was reduced by 91 and 98%, respectively, (both P

Atherosclerosis 2001 Aug;157(2):375-81 Related Articles, Books, LinkOut Dietary vegetable oil and wood derived plant stanol esters reduce atherosclerotic lesion size and severity in apoE*3-Leiden transgenic mice.Volger OL, Mensink RP, Plat J, Hornstra G, Havekes LM, Princen HM.Gaubius Lab. TNO-PG, PO Box 2215, 2301 CE Leiden, The Netherlands.The hypolipidemic and anti-atherosclerotic effects of vegetable oil- and wood-based dietary plant stanol esters were compared in female apoE*3-Leiden transgenic mice at relevant plasma cholesterol levels. The plant stanol esters derived from vegetable oil (sitostanol 65.7%, campestanol 30.1%) had different contents of sitostanol and campestanol than the plant stanol esters derived from wood (sitostanol 87.6%, campestanol 9.5%) or from a mixture of vegetable oil and wood (sitostanol 73.0%, campestanol 24.7%). The mice (10 per group) received for 38 weeks a control diet or diets containing 1.0% (w/w) plant stanol esters derived from either vegetable oil, wood or a mixture of both. Vegetable oil (-46%), wood (-42%) and vegetable oil/wood (-51%) plant stanol esters decreased the plasma cholesterol levels (P

The ability of triglyceride (TG)-rich lipoproteins from apolipoprotein (apo) E2 heterozygote, apo E3/2 which is relatively common, to stimulate cholesteryl ester synthesis in human monocyte-derived macrophages was studied to clarify the atherogenicity of apo E3/2. Twenty-three subjects were randomly sampled from our hospitals, and divided into the following 4 groups: 7 apo E3/3 subjects with normolipidemia, 6 apo E3/3 subjects with hypertriglyceridemia (HTG), 5 apo E3/2 subjects with normolipidemia and 5 apo E3/2 subjects with HTG. Plasma levels of TG and total cholesterol (chol) were significantly (P0.001) higher in apo E3/3 and apo E3/2 subjects with HTG than in apo E3/3 and apo E3/2 subjects with normolipidemia, but plasma levels of TG and total chol were not significantly different between apo E3/3 and apo E3/2 subjects with HTG. [14C]oleate incorporation into cholesteryl esters in macrophages was significantly (P0.001) higher in apo E3/2 subjects with HTG (0.661 nmol/mg cell protein) than in apo E3/3 subjects with normolipidemia (0.228) and with HTG (0.325) and in apo E3/2 subjects with normolipidemia (0.257). It is concluded that TG-rich lipoproteins from apolipoprotein E3/2 subjects with HTG enhance cholesteryl ester synthesis in human macrophages.

Postprandial fat clearance and absorption, fecal elimination and synthesis of cholesterol, bile acid synthesis, and cholesterol precursors and plant sterols in serum were studied in five patients with type III dyslipoproteinemia off and on lovastatin. The basal values were related to those in nontreated normolipidemic control subjects with apolipoprotein E3/3 phenotype (apo E3 controls, n = 16). On regular home diets, cholesterol precursor concentrations and cholesterol precursor/cholesterol ratios were high in the type III group. However, cholesterol absorption efficiency, bile acid and cholesterol synthesis measured with sterol balance technique and the precursor sterol/plant sterol ratios in serum were similar to the control values, suggesting that cholesterol absorption and metabolism was normal in these subjects. Lovastatin normalized the increased lipoprotein concentrations and reduced biliary cholesterol secretion, absolute absorption of cholesterol, precursor sterol/cholesterol and precursor sterol/plant sterol ratios in serum, fecal neutral and total sterol outputs and cholesterol synthesis. Lovastatin had no effect on cholesterol absorption efficiency or bile acid synthesis. Despite normalization of the triglyceride-rich lipoprotein levels by lovastatin, the postprandial vitamin A and squalene peak concentrations and the areas under the curves remained above the control ranges. The findings show that in type III hyperlipidemia, the precursor sterol/cholesterol ratios do not predict cholesterol synthesis. The latter, bile acid synthesis, precursor sterol/plant sterol ratios in serum, and cholesterol absorption are normal under basal conditions. The normalization of increased lipids by lovastatin is mainly due to reduced synthesis and absolute absorption of cholesterol, while the retarded postprandial fat clearance was not normalized by the drug.

HDL has been shown to enhance the removal of cholesterol from cultured fibroblasts, smooth muscle cells and macrophages, but fails to stimulate cholesterol removal from J-774 macrophages. Since J-774 macrophages do not synthesize or secrete apolipoprotein E, the effect of exogenous apolipoprotein E on HDL-mediated cellular cholesterol efflux was studied in this cell line. In cholesterol loaded J-774 macrophages total cellular cholesterol increased up to 6–7-fold, mainly cholesteryl esters. HDL 3 removed up to 30% of total cellular cholesterol with a decrease in cholesteryl ester levels while free cholesterol levels remained unchanged. HDL 3 was slightly superior to albumin in promoting cellular cholesterol removal. Exogenous apo E, over a wide range of apo E concentrations, did not enhance the ability of HDL 3 to remove cellular cholesterol from cholesterol loaded J-774 cells. Exogenous apo E did not promote HDL-mediated cholesterol efflux from cells, thus suggesting a possible role for the intracellular route of newly synthesized apo E in these processes.

Serum squalene, a non-steroid intermediate of cholesterol biosynthesis, originates mainly from endogenous cholesterol synthesis and partly from diet, especially in populations consuming a lot of olive oil rich in squalene. Its postabsorptive metabolism has not been studied in detail in humans. Its presence in chylomicrons and VLDL suggests that the removal of dietary squalene may reflect the metabolism of intestinal lipoproteins. Accordingly, we studied the postabsorptive metabolism of 1 g dietary squalene in 16 healthy subjects with apolipoprotein (apo) E 3/3 phenotype and in five type III hyperlipidemic apo E 2/2 homozygotes known to have a retarded chylomicron remnant removal, and compared the results with vitamin A fat load test. About 40% of the basal and 90% of the postabsorptive squalene was in lipoproteins < 1.019 g/ml. The peak concentrations of chylomicron squalene were at 6 h, and of triglyceride-rich nonchylo-fraction at 9-12 h in the controls. The peak values occurred later than those of vitamin A. At 24 h the levels still exceeded the basal ones. In type III dyslipoproteinemia, most of the basal and postabsorptive squalene was in lipoproteins of density less than 1.019 g/ml, the peak postabsorptive values occurred later than in the controls and the serum values remained above the control levels for up to 24 h. The squalene and vitamin A areas under the incremental response curves (AUC) were higher than in the control group. The AUCs of the two markers in chylomicron were correlated negatively and those in LDL+HDL were correlated positively with fasting HDL cholesterol levels, the respective correlations being opposite with fasting VLDL triglycerides. The postabsorptive profile of squalene levels resembled that of vitamin A in both groups, except that the squalene curves were shifted to a later time period. Thus, a delayed clearance of chylomicron remnants could be detected by analyzing serum squalene 6-24 h after the squalene-supplemented fat meal.

Apolipoprotein (apo) E phenotype was examined in 188 healthy subjects and in 447 patients seen between 1984 and 1986. The frequency of the apo E2, 3, and 4 genes in the clinically healthy subjects was 0.035 +/- 0.0288, 0.872 +/- 0.0310, and 0.093 +/- 0.0152, respectively. The frequency of the apo E3 gene was higher and that of the apo E genes 2 and 4 lower than that reported in western countries. Clinical features and apo E phenotype are presented from the 5 patients with type III hyperlipoproteinemia (HLP) due to apo E phenotype E2/2 (E2-III); all patients in E2-III were post-menopausal women. In contrast to the clinical characteristics so far reported, no notable findings of atherosclerosis, such as coronary angiographic findings or xanthoma, were evident in any of these 5 patients. Glucose intolerance was seen in 4 of them. Four patients were normolipidemic with apo E phenotype E2/2 (E2-N). In addition, plasma lipid and apolipoprotein concentrations were determined in patients with different apo E phenotypes. Plasma total cholesterol (TC) and apo B levels were elevated in the order of E2-N, E2/3, E2/4, E3/3, E3/4 and E4/4 except for E2-III. The plasma apo E level was highest in E2-III but was not significantly different from other phenotypes. The apo B/apo E and apo C-III/apo E ratios were significantly lower in E2/2 than in other phenotypes. The TC/apo B ratio was significantly higher in E2/2 than in other phenotypes.