bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of Apolipoprotein E3 in Transgenic Rabbits Causes Combined Hyperlipidemia by Stimulating Hepatic VLDL Production and Impairing VLDL Lipolysis
Authors: Huang Yadong , Ji Zhong-Sheng , Brecht Walter J. , Rall Stanley C. , Taylor John M. , Mahley Robert W.
Published: June 2011

Abstract
—The differential effects of overexpression of human apolipoprotein (apo) E3 on plasma cholesterol and triglyceride metabolism were investigated in transgenic rabbits expressing low (<10 mg/dL), medium (10 to 20 mg/dL), or high (>20 mg/dL) levels of apoE3. Cholesterol levels increased progressively with increasing levels of apoE3, whereas triglyceride levels were not significantly affected at apoE3 levels up to 20 mg/dL but were markedly increased at levels of apoE3 >20 mg/dL. The medium expressers had marked hypercholesterolemia (up to 3- to 4-fold over nontransgenics), characterized by an increase in low density lipoprotein (LDL) cholesterol, while the low expressers had only slightly increased plasma cholesterol levels. The medium expressers displayed an 18-fold increase in LDL but also had a 2-fold increase in hepatic very low density lipoprotein (VLDL) triglyceride production, an 8-fold increase in VLDL apoB, and a moderate decrease in the ability of the VLDL to be lipolyzed. However, plasma clearance of VLDL was increased, likely because of the increased apoE3 content. The increase in LDL appears to be due to an enhanced competition of VLDL for LDL receptor binding and uptake, resulting in the accumulation of LDL. The combined hyperlipidemia of the apoE3 high expressers (>20 mg/dL) was characterized by a 19-fold increase in LDL cholesterol but also a 4-fold increase in hepatic VLDL triglyceride production associated with a marked elevation of plasma VLDL triglycerides, cholesterol, and apoB100 (4-, 9-, and 25-fold over nontransgenics, respectively). The VLDL from the high expressers was much more enriched in apoE3 and markedly depleted in apoC-II, which contributed to a >60% inhibition of VLDL lipolysis. The combined effects of stimulated VLDL production and impaired VLDL lipolysis accounted for the increases in plasma triglyceride and VLDL concentrations in the apoE3 high expressers. The hyperlipidemic apoE3 rabbits have phenotypes similar to those of familial combined hyperlipidemia, in which VLDL overproduction is a major biochemical feature. Overall, elevated expression of apoE3 appears to determine plasma lipid levels by stimulating hepatic VLDL production, enhancing VLDL clearance, and inhibiting VLDL lipolysis. Thus, the differential expression of apoE may, within a rather narrow range of concentrations, play a critical role in modulating plasma cholesterol and triglyceride levels and may represent an important determinant of specific types of hyperlipoproteinemia.
Pattern of Alcohol Drinking and Progression of Atherosclerosis
Authors: Kauhanen Jussi , Kaplan George A. , Goldberg Debbie E. , Salonen Riitta , Salonen Jukka T.
Published: June 2011

Abstract
—Most studies that examine the role of alcohol consumption in atherosclerosis and cardiovascular disease have overlooked the possible effect of drinking pattern. We investigated the association between the habit of heavy acute intake of beer and spirits (binging) and the 4-year progression of carotid atherosclerosis in a population-based sample of middle-aged Finnish men. Data from the Kuopio Ischemic Heart Disease Risk Factor Study (KIHD) were used to estimate changes in maximum and mean intima-media thickness (IMT) and the maximum plaque height in 764 KIHD participants who reported using beer and in 871 participants who used spirits. After adjustment for age, baseline carotid atherosclerosis, and average weekly alcohol consumption level, we observed the highest atherosclerosis progression in men who usually consumed a whole bottle of vodka or more in 1 session. For beer binging (>6 beers at a time), the magnitude of IMT progression was even higher, although this association was only marginally significant (
P
<0.1) because of smaller numbers. The associations were largely unaffected by adjustments for blood pressure, lipids, smoking, BMI, and medication. The magnitude of the difference was generally higher in a subgroup that was free of IHD at baseline. We conclude that the pattern of drinking associates with the progression of carotid atherosclerosis independently of the total level of alcohol consumption and risk factors.
Activation of Protein Kinase C Is Required for the Stable Attachment of Adherent Platelets to Collagen but Is Not Needed for the Initial Rapid Adhesion Under Flow Conditions
Authors: Polanowska-Grabowska Renata , Gear Adrian R. L.
Published: June 2011

Abstract—We have investigated the role of protein kinase C (PKC) in the initial events of α2β1-integrin–mediated platelet adhesion to collagen under flow conditions. Although adhesion caused activation of PKC, as evidenced by pleckstrin phosphorylation, the PKC inhibitors GF 109203X and Gö 6976 had no effect on adhesion, even though they prevented pleckstrin phosphorylation. The initial kinetics and extent of platelet adhesion to collagen (<5 seconds) and tyrosine phosphorylation of p125FAKand p72sykwere not influenced by the PKC inhibitors, whereas adhesion to polylysine was prevented. These results indicate that adhesion to collagen and polylysine involve different mechanisms and requirements for PKC activation. Pretreatment with GF 109203X destabilized collagen-adherent platelets, accelerating their detachment, which was associated with tyrosine dephosphorylation of p125FAK. Thus, although PKC activation was not required for rapid platelet adhesion to collagen, it appears to play an important role in stabilizing the attachment of adherent platelets to collagen. We also examined the effect of PKC activation by the phorbol ester phorbol 12-myristate 13-acetate (PMA) on platelet adhesion to collagen. PMA at 100 nmol/L strongly potentiated adhesion and tyrosine phosphorylation of p125FAKand p72sykand activated β1-integrins, as determined by increased exposure of the 15/7 epitope. The PMA-stimulated adhesion was partially blocked by an anti-α2β1antibody, was completely inhibited by GF 109203X, and was not correlated with the extent of pleckstrin phosphorylation. Therefore, strong PKC activation may lead to inside-out signaling, enhancing the role of β1-integrins in adhesion. Pleckstrin phosphorylation does not appear to be involved in the initial phase of basic or PMA-stimulated adhesion but may help stabilize the adherent platelets.
Two-Chain Factor VIIa Generated in the Pericardium During Surgery With Cardiopulmonary Bypass
Authors: Philippou Helen , Davidson Simon J. , Mole M. Teresa , Pepper John R. , Burman John F. , Lane David A.
Published: June 2011

Abstract
—Several recent studies have proposed that coagulation is triggered during cardiopulmonary bypass surgery by extrinsic pathway activation involving factor VIIa generation, but the methodology was indirect. Therefore, 12 patients were studied during routine cardiac and cardiopulmonary bypass surgery. Samples were taken before, during, and after bypass from the perfusate, from the aorta (retrograde cardiac drainage), pericardium, and collected suction fluid originating from the whole operative field. These samples were analyzed by enzyme-linked immunosorbent assay for 2-chain factor VIIa, by prothrombin F
1+2
assay, by thrombin-antithrombin (TAT) assay, and for heparin concentration. Factor VIIa, F
1+2
, and TAT levels in samples from the pericardium were greatly elevated (mean, 0.92 to 1.01, 227 to 334, and 399 to 526 μg/L, respectively; preoperative mean, 0.33, 32.3, and 1.90 μg/L, respectively;
P
<0.05 for all), whereas levels in suction fluid were less consistently high. Factor VIIa and both F
1+2
and thrombin-antithrombin levels in samples from the aorta, pericardium, and suction fluid were significantly correlated (
r
=0.57,
P
<0.001, n=111; and
r
=0.51,
P
<0.001, n=105, respectively), and all were inversely correlated with heparin levels (
r
>−0.35,
P
<0.001, n>92). There was no evidence of factor VIIa generation in the circuit during bypass surgery, and both F
1+2
and thrombin-antithrombin levels rose only ≈2-fold, probably because heparin levels were higher than they were in the pericardium (
P
<0.05). We concluded that appreciable activation of factor VII occurs on the pericardium and that this is associated with increased thrombin generation. Ineffective local heparinization may be partly responsible. These results suggest that pericardium-induced activation of factor VII should be the target of anticoagulant strategies during cardiopulmonary bypass surgery.
Cholesterol or Triglyceride Loading of Human Monocyte-Derived Macrophages by Incubation With Modified Lipoproteins Does Not Induce Tissue Factor Expression
Authors: van den Eijnden Mark M. E. D. , van Noort Jacqueline T. , Hollaar Leny , van der Laarse Arnoud , Bertina Rogier M.
Published: June 2011

Abstract—Macrophages/foam cells localized in cholesterol- and triglyceride-rich regions of atherosclerotic plaques express high levels of tissue factor (TF), the essential cofactor and receptor of factor VIIa. It is not clear whether modified lipoproteins, for which several agonistic effects on macrophages have been described, are independent stimuli of TF expression in these cells. Therefore, we studied the effect of short-term (1 day) and long-term (4 to 7 days) incubation of human monocyte-derived macrophages cultured in suspension with modified and native LDLs or VLDLs on the expression of TF mRNA, antigen, and activity. We used native LDL or VLDL, moderately oxidized LDL or VLDL, severely oxidized LDL or VLDL, acetylated LDL, and β-VLDL at a protein concentration of 100 μg/mL. Cholesterol loading occurred within 9 hours after the addition of acetylated LDL and continued during long-term incubation. Incubation of severely oxidized LDL for 7 days resulted in a slight increase in cholesterol content. Triglyceride loading was observed during short-term and long-term incubation with native and modified VLDLs. Neither cholesterol nor triglyceride loading resulted in expression of TF. Bacterial LPS still could induce TF expression in lipid-laden macrophages. Our results show that incubation with modified lipoproteins or lipid loading does not lead to TF expression in monocyte-derived macrophages cultured in suspension. This suggests that induction of TF expression in foam cells in the atherosclerotic lesion is triggered by additional or other components.
Is the Response of Serum Lipids and Lipoproteins to Postmenopausal Hormone Replacement Therapy Modified by ApoE Genotype?
Authors: Heikkinen Anna-Mari , Niskanen Leo , Ryynänen Markku , Komulainen Marja H. , Tuppurainen Marjo T. , Parviainen Markku , Saarikoski Seppo
Published: June 2011

Abstract
—Postmenopausal hormone replacement therapy (HRT) has favorable effects on the serum lipid profile, and it also decreases the risk of cardiovascular diseases. The apolipoprotein E genotype has influence on serum levels of lipids and lipoproteins; apoE allele ε4 (apoE4) is associated with high total and LDL cholesterol levels. Genotype also influences the lipid responses to treatment with diet and statins, but the effect of HRT in different apoE genotypes is unknown. We studied the effects of HRT on the concentrations of serum lipids in apoE4-positive early postmenopausal women (genotypes 3/4 and 4/4) compared with apoE4-negative women (genotypes 2/3 and 3/3) in a population-based, prospective 5-year study. In all, 232 early postmenopausal women were randomized into 2 treatment groups: an HRT group (n=116), which received a sequential combination of 2 mg estradiol valerate (E
2
Val) from day 1 to 21 and 1 mg cyproterone acetate (CPA) from day 12 to 21 (Climen), and a placebo group (n=116), which received 500 mg/d calcium lactate. Serum concentrations of total, LDL, and HDL cholesterol and triglycerides were measured at baseline and after 2 and 5 years of treatment. A total of 154 women completed the final analysis. During the follow-up period, serum total cholesterol and LDL cholesterol concentrations decreased in the HRT group in apoE4-negative women (8.1% and 17.1%, respectively;
P
<0.001) but did not change in the HRT group in apoE4-positive women or in the placebo group. Serum HDL cholesterol concentrations decreased in the placebo group (apoE4-negative, 3.9%,
P
=0.015; apoE4-positive, 8.1%,
P
=0.004) but did not change significantly in the HRT group. Serum triglyceride levels tended to increase in both study groups and genotypes (15.1% to 36.2%,
P
<0.038 to 0.001), but no differences were observed between the study groups or genotypes, respectively. Our finding was that in postmenopausal Finnish women LDL cholesterol levels in apoE4-negative subjects respond more favorably to HRT than those in apoE4-positive subjects. This finding has potential importance in postmenopausal women with hypercholesterolemia, if confirmed in other studies.
Quantitative Trait Locus Analysis of Plasma Lipoprotein Levels in an Autoimmune Mouse Model
Authors: Gu Lingjie , Johnson Michael W. , Lusis Aldons J.
Published: June 2011

Abstract—The autoimmune MRL/lprmouse strain, a model for systemic lupus erythematosus, exhibited an unusual plasma lipoprotein profile, suggesting a possible interaction of autoimmune disease and lipoprotein metabolism. In an effort to examine the genetic basis of such interactions, and to study their relationship to atherogenesis, we performed a quantitative trait locus analysis using a total of 272 (MRL/lpr×BALB/cJ) second generation (F2) intercross mice. These mice were examined for levels of total plasma cholesterol, HDL cholesterol, VLDL and LDL cholesterol, unesterified cholesterol, autoantibodies, and aortic fatty streak lesions. Using a genome scan approach, we identified 4 quantitative trait loci controlling plasma lipoprotein levels on chromosomes (Chrs) 5, 8, 15, and 19. The locus on Chr 15 exhibited lod scores of 11.1 for total cholesterol and 6.7 for VLDL and LDL cholesterol in mice fed an atherogenic diet, and it contains a candidate gene, the sterol regulatory element binding protein-2. The locus on Chr 5 exhibited lod scores of 3.8 for total cholesterol and 4.1 for unesterified cholesterol in mice fed an atherogenic diet, and this locus has been observed in 2 previous studies. The locus on Chr 8 exhibited a lod score of 3.1 for unesterified cholesterol in mice fed a chow diet. This locus contains the lecithin-cholesterol acyltransferase gene, and decreased activity of the enzyme in the MRL strain suggests that this gene underlies the quantitative-trait locus. The locus on Chr 19 exhibited a lod score of 8.4 for HDL cholesterol and includes theFasgene, which is mutated in MRL/lprmice and is primarily responsible for the autoimmune phenotype in this cross. That theFasgene is responsible for the HDL quantitative-trait loci is supported by the finding that autoantibody levels were strongly correlated with HDL cholesterol levels (ρ=−0.37,P<0.0001) among the F2 mice. HDL cholesterol levels were in turn significantly associated with aortic fatty streak lesions among the F2 mice (ρ=−0.17,P=0.006). Further, there was a threshold effect of autoantibody levels on the development of fatty streak lesions (ρ=0.45,P=0.004 for 42 F2 mice with anti-dsDNA Ab over 0.5 OD). Our results support the concept that the high prevalence of coronary artery disease in systemic lupus erythematosus is due in part to a reduction of HDL cholesterol levels resulting from the autoimmune disease.
Binding of β-VLDL to Heparan Sulfate Proteoglycans Requires Lipoprotein Lipase, Whereas ApoE Only Modulates Binding Affinity
Authors: de Beer Femke , Hendriks Wendy L. , van Vark Leonie C. , Kamerling Sylvia W.A. , van Dijk Ko Willems , Hofker Marten H. , Smelt Augustinus H.M. , Havekes Louis M.
Published: June 2011

Abstract
—The binding of β-VLDL to heparan sulfate proteoglycans (HSPG) has been reported to be stimulated by both apoE and lipoprotein lipase (LPL). In the present study we investigated the effect of the isoform and the amount of apoE per particle, as well as the role of LPL on the binding of β-VLDL to HSPG. Therefore, we isolated β-VLDL from transgenic mice, expressing either APOE*2(Arg158→Cys) or APOE*3-Leiden (E2-VLDL and E3Leiden-VLDL, respectively), as well as from apoE-deficient mice containing no apoE at all (Enull-VLDL). In the absence of LPL, the binding affinity and maximal binding capacity of all β-VLDL samples for HSPG-coated microtiter plates was very low. Addition of LPL to this cell-free system resulted in a 12- to 55-fold increase in the binding affinity and a 7- to 15-fold increase in the maximal binding capacity (
B
max
). In the presence of LPL, the association constant (
K
a
) tended to increase in the order Enull-VLDL<E2-VLDL<E3Leiden-VLDL, whereas
B
max
increased in the reverse order: E3Leiden-VLDL≈E2-VLDL<Enull-VLDL. Addition of LPL resulted in a marked stimulation of both
K
a
and
B
max
for binding of β-VLDL samples to J774 cells similar to that found for the binding to HSPG-LPL complexes. Our results indicate that both
K
a
and
B
max
for binding of β-VLDL to HSPG are increased more than 1 order of magnitude on addition of LPL. In addition, for the binding of β-VLDL to HSPG-LPL complexes, the presence of apoE is not a prerequisite, but results in an increased binding affinity, depending on the apoE isoform used.
Upregulation of Superoxide Dismutase and Nitric Oxide Synthase Mediates the Apoptosis-Suppressive Effects of Shear Stress on Endothelial Cells
Authors: Dimmeler Stefanie , Hermann Corinna , Galle Jan , Zeiher Andreas M.
Published: June 2011

Abstract
—Physiological levels of laminar shear stress completely abrogate apoptosis of human endothelial cells in response to a variety of stimuli and might therefore importantly contribute to endothelial integrity. We show here that the apoptosis-suppressive effects of shear stress are mediated by upregulation of Cu/Zn SOD and NO synthase. Shear stress-mediated inhibition of endothelial cell apoptosis in response to exogenous oxygen radicals, oxidized LDL, and tumor necrosis factor-α was associated with complete inhibition of caspase-3-like activity, the central effector arm executing the apoptotic cell death program in endothelial cells. Shear stress-dependent upregulation of Cu/Zn SOD and NO synthase blocks activation of the caspase cascade in response to apoptosis-inducing stimuli. These findings establish the upregulation of Cu/Zn SOD and NO synthase by shear stress as a central protective cellular mechanism to preserve the integrity of the endothelium after proapoptotic stimulation.
Reduced In Vitro Repair In Endothelial Cells Harvested From the Intercostal Ostia of Porcine Thoracic Aorta
Authors: Akong Timothy A. , Gotlieb Avrum I.
Published: June 2011

Abstract—The ability of large-vessel endothelium to repair itself rapidly after injury is important in the maintenance of its barrier function and in limiting the development and progression of atherosclerosis. Because dysfunctional repair may be involved in the pathogenesis of some atherosclerotic plaques, including those at the ostia of aortic branches, linear mechanical denuding wounds were made in confluent monolayers of endothelial cells harvested by scraping from the flow divider, the upstream wall of the intercostal branch and unbranched regions in the thoracic aorta. The extent of wound closure was significantly lower in cells derived from either side of the intercostal branches, compared with cells from unbranched areas. The wound edge of cells harvested from the flow divider and its opposite wall closed by 22±0.084 μm and 22±1.3 μm, respectively, versus control, unbranched endothelial cells (30±2.2 μm) at 24 hours and by 48 hours, 48±3.4 μm and 47±3.6 μm compared with control (61±3.4 μm). Extent of wound closure in cells harvested by scraping from unbranched regions was comparable with collagenase-harvested endothelial cells at 24 and 48 hours. Distribution of F-actin microfilaments, tubulin and centrosomes have been shown to be disrupted at the wound edge in poorly migrating cells. In our study, however, no differences were observed in cytoskeletal distribution between cells from branched, unbranched and control areas. Thus, aortic endothelial cells from the intercostal branch region show a reduced ability to repair wounds compared with cells harvested from unbranched aorta. The mechanism for this difference is currently unknown.
Lipid Transfer Inhibitor Protein Defines the Participation of Lipoproteins in Lipid Transfer Reactions
Authors: Serdyuk Anatole P. , Morton Richard E.
Published: June 2011

Abstract
—Cholesteryl ester transfer protein (CETP) catalyzes the net transfer of cholesteryl ester (CE) between lipoproteins in exchange for triglyceride (heteroexchange). It is generally held that CETP primarily associates with HDL and preferentially transfers lipids from this lipoprotein fraction. This is illustrated in normal plasma where HDL is the primary donor of the CE transferred to VLDL by CETP. However, in plasma deficient in lipid transfer inhibitor protein (LTIP) activity, HDL and LDL are equivalent donors of CE to VLDL (
Arterioscler Thromb Vasc Biol
. 1997;17:1716–1724). Thus, we have hypothesized that the preferential transfer of CE from HDL in normal plasma is a consequence of LTIP activity and not caused by a preferential CETP-HDL interaction. We have tested this hypothesis in lipid mass transfer assays with partially purified CETP and LTIP, and isolated lipoproteins. With a physiological mixture of lipoproteins, the preference ratio (PR, ratio of CE mass transferred from a lipoprotein to VLDL versus its CE content) for HDL and LDL in the presence of CETP alone was ≈1 (ie, no preference). Fourfold variations in the LDL/HDL ratio or in the levels of HDL in the assay did not result in significant preferential transfer from any lipoprotein. On addition of LTIP, the PR for HDL was increased up to 2-fold and that for LDL decreased in a concentration-dependent manner. Under all conditions where LDL and HDL levels were varied, LTIP consistently resulted in a PR >1 for CE transfer from HDL. Short-term experiments with radiolabeled lipoproteins and either partially purified or homogenous CETP confirmed these observations and further demonstrated that CETP has a strong predilection to mediate homoexchange (bidirectional transfer of the same lipid) rather than heteroexchange (CE for TG); LTIP had no effect on the selection of CE or TG by CETP or its mechanism of action. We conclude, in contrast to current opinion, that CETP has no preference for CE in HDL versus LDL, suggesting that the previously reported stable binding of CETP to HDL does not result in selective transfer from this lipoprotein. These data suggest that LTIP is responsible for the preferential transfer of CE from HDL that occurs in plasma. CETP and LTIP cooperatively determine the extent of CETP-mediated remodeling of individual lipoprotein fractions.
Role of Cholesterol Ester Mass in Regulation of Secretion of ApoB100 Lipoprotein Particles by Hamster Hepatocytes and Effects of Statins on That Relationship
Authors: Zhang Zujun , Cianflone Katherine , Sniderman Allan D.
Published: June 2011

Abstract
—Our understanding of the factors that regulate the secretion of apoB100 lipoproteins remains incomplete with considerable debate as to the role, if any, for cholesterol ester in this process. This study examines this issue in primary cultures of hamster hepatocytes, a species in which both cholesterol and apoB100 metabolism are very similar to man. Addition of oleate to medium increased the mass of triglyceride and cholesterol ester within the hepatocyte and also increased the secretion of triglycerides, cholesterol ester, and apoB100 into the medium. Next, the responses of hamster hepatocytes to addition of either an HMG-CoA reductase inhibitor (lovastatin) or an acyl-CoA cholesterol acyltransferase inhibitor (58-035) to the medium, with or without added oleate, were determined. Effects of either agent were only evident in the oleate-supplemented medium in which cholesterol ester mass had been increased above basal. If oleate was not added to the medium, neither agent reduced apoB100 secretion; equally important, over the 24-hour incubation, neither agent, at the concentration used, produced any detectable change in intracellular cholesterol ester mass. However, in contrast to the estimates of mass, which were unchanged, under the same conditions radioisotopic estimates of cholesterol ester synthesis were markedly reduced. Any conclusion as to the relation of cholesterol ester mass to apoB100 secretion would therefore depend on which of the 2 methods was used. Overall, the data indicate a close correlation between the mass of cholesterol ester within the hepatocyte and apoB100 secretion from it and they go far to explain previous apparently contradictory data as to this relation. More importantly, though, taken with other available data, they indicate that the primary response of the liver to increased delivery of lipid is increased secretion rather than decreased uptake. These results point, therefore, to a hierarchy of hepatic responses to increased flux of fatty acids and increased synthesis of cholesterol that in turn suggests a more dynamic model of cholesterol homeostasis in the liver than has been appreciated in the past.
A Genome Search Identifies Major Quantitative Trait Loci on Human Chromosomes 3 and 4 That Influence Cholesterol Concentrations in Small LDL Particles
Authors: Rainwater David L. , Almasy Laura , Blangero John , Cole Shelley A. , VandeBerg John L. , MacCluer Jean W. , Hixson James E.
Published: June 2011

Abstract
—Small, dense LDL particles are associated with increased risk of cardiovascular disease. To identify the genes that influence LDL size variation, we performed a genome-wide screen for cholesterol concentrations in 4 LDL size fractions. Samples from 470 members of randomly ascertained families were typed for 331 microsatellite markers spaced at ≈15 cM intervals. Plasma LDLs were resolved by using nondenaturing gradient gel electrophoresis into 4 fraction sizes (LDL-1, 26.4 to 29.0 nm; LDL-2, 25.5 to 26.4 nm; LDL-3, 24.2 to 25.5 nm; and LDL-4, 21.0 to 24.2 nm) and cholesterol concentrations were estimated by staining with Sudan Black B. Linkage analyses used variance component methods that exploited all of the genotypic and phenotypic information in the large extended pedigrees. In multipoint linkage analyses with quantitative trait loci for the 4 fraction sizes, only LDL-3, a fraction containing small LDL particles, gave peak multipoint log
10
odds in favor of linkage (LOD) scores that exceeded 3.0, a nominal criterion for evidence of significant linkage. The highest LOD scores for LDL-3 were found on chromosomes 3 (LOD=4.1), 4 (LOD=4.1), and 6 (LOD=2.9). In oligogenic analyses, the 2-locus LOD score (for chromosomes 3 and 4) increased significantly (
P
=0.0012) to 6.1, but including the third locus on chromosome 6 did not significantly improve the LOD score (
P
=0.064). Thus, we have localized 2 major quantitative trait loci that influence variation in cholesterol concentrations of small LDL particles. The 2 quantitative trait loci on chromosomes 3 and 4 are located in regions that contain the genes for apoD and the large subunit of the microsomal triglyceride transfer protein, respectively.
Ultrastructure of Early Lipid Accumulation in ApoE-Deficient Mice
Authors: Tamminen M. , Mottino G. , Qiao J. H. , Breslow J. L. , Frank J. S.
Published: June 2011

Abstract
—Apolipoprotein (apo) E–deficient mice develop severe hypercholesterolemia and have lesions that progress from fatty streaks to fibrous plaques distributed in lesion-prone areas throughout the aorta. Lesions develop in apoE-deficient mice on a regular chow diet and will occur faster on a diet higher in cholesterol. Examination of the aortas from these mice on a chow diet by high-resolution, freeze-etch electron microscopy demonstrated lipid retention in the intima by 3 weeks of age. Lipid was retained in the matrix as individual particles between 33 and 48 nm in diameter, aligned along the collagen fibrils and in aggregates consisting of lipid particles with average diameters of 33 and 68 nm. Larger particles seemed to have formed from fusion of smaller particles. Lipid retention was more widespread in 5- and 9-week-old mice. Monocyte attachment to endothelial cells was observed by electron microscopy at 5 weeks of age. The appearance of the intimal lipid was similar to that previously described in rabbit models and suggests that lipid interaction with matrix filaments and subsequent aggregation of lipid particles are critical first steps in the process of foam cell formation.
Hormone Replacement Therapy, Inflammation, and Hemostasis in Elderly Women
Authors: Cushman Mary , Meilahn Elaine N. , Psaty Bruce M. , Kuller Lewis H. , Dobs Adrian S. , Tracy Russell P.
Published: June 2011

Abstract
—Lipid-lowering by postmenopausal hormone therapy (HRT) explains only partly the assumed coronary risk reduction associated with therapy. To explore other possible mechanisms, we studied associations of HRT use with inflammation and hemostasis risk markers in women ≥65 years of age. Subjects were selected from 3393 participants in the fourth year examination of the Cardiovascular Health Study, an observational study of vascular disease risk factors. After excluding women with vascular disease, we compared levels of inflammation and hemostasis variables in the 230 women using unopposed estrogen and 60 using estrogen/progestin, with those of 196 nonusers selected as controls. Compared with nonusers, unopposed estrogen use was associated with 59% higher mean C-reactive protein (
P
<0.001), but with modestly lower levels of other inflammation indicators, fibrinogen, and alpha-1 acid glycoprotein (
P
<0.001). Factor VIIc was 16% higher among estrogen users (
P
<0.001), but this was not associated with higher thrombin production (prothrombin fragment 1-2), or increased fibrin breakdown (D-dimer). Concentration of plasminogen activator inhibitor-1 was 50% lower in both using groups (
P
<0.001) compared with nonusers, and this was associated with higher plasmin-antiplasmin complex: 8% higher in estrogen and 18% higher in estrogen/progestin users (
P
<0.05). Relationships between the markers and hormone use were less pronounced in estrogen/progestin users, with no association for C-reactive protein except in women in upper 2 tertiles of body mass index (
P
for interaction, 0.02). The direction and strength of the associations of HRT use with inflammation markers differed depending on the protein, so it is not clear whether HRT confers coronary risk reduction through an inflammation-sensitive mechanism. Associations with hemostasis markers indicated no association with evidence of procoagulation and a possible association with increased fibrinolytic activity.
C-Reactive Protein in Healthy Subjects: Associations With Obesity, Insulin Resistance, and Endothelial Dysfunction
Authors: Yudkin John S. , Stehouwer C. D. A. , Emeis J. J. , Coppack S. W.
Published: June 2011

Abstract
—C-reactive protein, a hepatic acute phase protein largely regulated by circulating levels of interleukin-6, predicts coronary heart disease incidence in healthy subjects. We have shown that subcutaneous adipose tissue secretes interleukin-6 in vivo. In this study we have sought associations of levels of C-reactive protein and interleukin-6 with measures of obesity and of chronic infection as their putative determinants. We have also related levels of C-reactive protein and interleukin-6 to markers of the insulin resistance syndrome and of endothelial dysfunction. We performed a cross-sectional study in 107 nondiabetic subjects: (1) Levels of C-reactive protein, and concentrations of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, were related to all measures of obesity, but titers of antibodies to
Helicobacter pylori
were only weakly and those of
Chlamydia pneumoniae
and cytomegalovirus were not significantly correlated with levels of these molecules. Levels of C-reactive protein were significantly related to those of interleukin-6 (
r
=0.37,
P
<0.0005) and tumor necrosis factor-α (
r
=0.46,
P
<0.0001). (2) Concentrations of C-reactive protein were related to insulin resistance as calculated from the homoeostasis model assessment model, blood pressure, HDL, and triglyceride, and to markers of endothelial dysfunction (plasma levels of von Willebrand factor, tissue plasminogen activator, and cellular fibronectin). A mean standard deviation score of levels of acute phase markers correlated closely with a similar score of insulin resistance syndrome variables (
r
=0.59,
P
<0.00005), this relationship being weakened only marginally by removing measures of obesity from the insulin resistance score (
r
=0.53,
P
<0.00005). These data suggest that adipose tissue is an important determinant of a low level, chronic inflammatory state as reflected by levels of interleukin-6, tumor necrosis factor-α, and C-reactive protein, and that infection with
H
pylori
,
C pneumoniae
, and cytomegalovirus is not. Moreover, our data support the concept that such a low-level, chronic inflammatory state may induce insulin resistance and endothelial dysfunction and thus link the latter phenomena with obesity and cardiovascular disease.
Effect of Lp(a) on the Early Functional and Structural Changes of Atherosclerosis
Authors: Raitakari Olli T. , Adams Mark R. , Celermajer David S.
Published: June 2011

Abstract
—Epidemiologic studies have shown a significant relationship between elevated plasma levels of Lp(a) and increased risk of cardiovascular events; however, the mechanisms by which elevated Lp(a) levels produce this increased risk are not known. To test the hypothesis that high Lp(a) levels might contribute to the development of subclinical atherosclerosis, we examined the influence of Lp(a) levels on early functional and structural atherosclerotic vascular changes. Flow-mediated (endothelium-dependent) and nitrate-mediated (smooth muscle–dependent) arterial dilations were measured by high-resolution ultrasound in 241 normal healthy subjects (aged 15 to 69 years; 116 men). In addition, carotid artery intima-media thickness was measured by ultrasound in 71 subjects. Plasma Lp(a) was measured using a 2-sided immunoradiometric assay (cohort median, 10 mg/dL; interquartile range, 3.9 to 24.4 mg/dL). In these subjects, there were no significant relationships between Lp(a) and arterial endothelial function, smooth muscle responses, or carotid wall thickness (
P
>0.25). By contrast, other lipid risk factors, such as LDL-cholesterol and LDL-cholesterol/HDL-cholesterol ratio, were significantly correlated with abnormal arterial function and structure (
P
≤0.01). These data suggest that elevated Lp(a) levels do not confer cardiovascular risk by contributing to the early functional or structural changes of atherosclerosis.
Glucocorticoid Resistance Caused by Reduced Expression of the Glucocorticoid Receptor in Cells From Human Vascular Lesions
Authors: Bray Paula J. , Du Baoheng , Mejia Victor M. , Hao Steven C. , Deutsch Ezra , Fu Chenzhong , Wilson Robert C. , Hanauske-Abel Hartmut , McCaffrey Timothy A.
Published: June 2011

Abstract—Mechanisms that control the balance between cell proliferation and death are important in the development of vascular lesions. Rat primary smooth muscle cells were 80% inhibited by low microgram doses of hydrocortisone (HC) and 50% inhibited by nanogram concentrations of transforming growth factor-β1 (TGF-β1), although some lines acquired resistance in late passage. However, comparable doses of HC, or TGF-β1, failed to inhibit most human lesion-derived cell (LDC) lines. In sensitive LDC, HC (10 μg/mL) inhibited proliferation by up to 50%, with obvious apoptosis in some lines, and TGF-β1 inhibited proliferation by more than 90%. Collagen production, as measured by [3H]proline incorporation or RIA for type III pro-collagen, was either unaffected or increased in the LDCs by HC. These divergent responses between LDC lines were partially explained by the absence of the glucocorticoid receptor (GR) and heat shock protein 90 mRNA in 10 of 12 LDC lines, but the presence of the mineralocorticoid receptor and 11β-hydroxysteroid dehydrogenase type II. Western blot analysis confirmed the absence of the GR protein in cells lacking GR mRNA. Immunohistochemistry of human carotid lesions showed high levels of GR in the tunica media, but large areas lacking GR in the fibrous lesion. Considering the absence of the GR in most lines, the effects of HC may be elicited through the mineralocorticoid receptor. Functional resistance to the antiproliferative and antifibrotic effects of HC may contribute to excessive wound repair in atherosclerosis and restenosis.
Factors Underlying the Increase in Carotid Intima-Media Thickness in Borderline Hypertensives
Authors: Pauletto P. , Palatini P. , Da Ros S. , Pagliara V. , Santipolo N. , Baccillieri S. , Casiglia E. , Mormino P. , Pessina A. C.
Published: June 2011

Abstract
—To define the role played by various risk and behavioral factors in the increase of carotid intima-media thickness (IMT) observed in borderline hypertensives. Using B-mode ultrasonography, we compared 97 borderline hypertensives enrolled in the HARVEST study to 27 normotensive controls. Intima-media thickness was measured in the right and left common carotid artery, bulb, and internal carotid artery. Mean IMT (m-IMT), maximum IMT (M-IMT), the mean of M-IMT (M-MAX), and the prevalence of raised lesions (IMT>1 mm) were established. Compared to the controls, higher systolic BP, diastolic BP, mean arterial blood pressure levels and body mass index (BMI) were present in the borderline hypertensives, whereas age, smoking, physical activity, serum cholesterol, and triglycerides were similar. After adjusting for age, sex, heart rate, BMI, smoking, serum cholesterol, triglycerides, and physical activity, higher values of m-IMT and M-IMT were present in most carotid segments of borderline hypertensives compared with controls. After further adjustment for systolic BP and diastolic BP, differences were no longer significant. The adjusted M-MAX was 0.59±0.12 in borderline hypertensives compared with 0.50±0.10 in controls (
P
<0.001). After adjustment for systolic BP and diastolic BP it was 0.58±0.11 in borderline hypertensives compared with 0.50±0.12 in controls (
P
<0.005). In the various carotid segments, the prevalence of raised lesions was 1.2% in borderline hypertensives compared with 0.3% in controls (
P
<0.001). In the multivariate analysis m-IMT, M-IMT, and M-MAX were related to ambulatory mean arterial pressure, systolic BP and diastolic BP, serum cholesterol and triglycerides, BMI, age, and physical activity. Higher IMT values were found in subjects who were physically active than in those who were sedentary. In borderline hypertensives, an increase in IMT takes place not only in the common carotid artery but also in the bulb and the internal carotid segment. Blood pressure levels are a main determinant of m-IMT while the interaction of BP with other risk factors such as age and plasma lipids is more relevant for advanced intima-media thickening such as M-MAX.
Modification of Type III VLDL, Their Remnants, and VLDL From ApoE-Knockout Mice by
p
-Hydroxyphenylacetaldehyde, a Product of Myeloperoxidase Activity, Causes Marked Cholesteryl Ester Accumulation in Macrophages
Authors: Whitman Stewart C. , Hazen Stanley L. , Miller David B. , Hegele Robert A. , Heinecke Jay W. , Huff Murray W.
Published: June 2011

Abstract
—Very low density lipoproteins (VLDLs) from apolipoprotein (apo) E2/E2 subjects with type III hyperlipoproteinemia, VLDL remnants, and VLDL from apoE-knockout (EKO) mice are taken up poorly by macrophages. The present study examined whether VLDL modification by the reactive aldehyde
p
-hydroxyphenylacetaldehyde (pHA) enhances cholesteryl ester (CE) accumulation by J774A.1 macrophages. pHA is the major product derived from the oxidation of
l
-tyrosine by myeloperoxidase and is a component of human atherosclerotic lesions. Incubation of J774A.1 cells with native type III VLDL, their remnants, and EKO-VLDL increased cellular CE by only 3-, 5-, and 5-fold, respectively, compared with controls. In striking contrast, cells exposed to VLDL modified by purified pHA (pHA-VLDL) exhibited marked increases in cellular CE of 38-, 47-, and 35-fold, respectively (
P
≤0.0001). Addition of the lipoprotein lipase inhibitor tetrahydrolipstatin decreased cellular CE accumulation induced by the 3 pHA-modified VLDL preparations by 73%, 59%, and 73%, respectively. Addition of the acyl coenzyme A:cholesterol acyltransferase inhibitor DuP 128 to cells incubated with the pHA-modified lipoproteins decreased cellular CE by 100%, 82%, and 95%, respectively, but had no effect on cellular triglycerides. To examine whether the type A scavenger receptors (SR-As) mediated the uptake of pHA-VLDL, incubations were performed in the presence of polyinosine (poly I), a polynucleotide known to block binding to SR-As (types I and II), or in cells preincubated with interferon-γ (IFN-γ), a cytokine known to decrease expression of SR-A type I. Coincubation of pHA-VLDL with poly I reduced cellular CE by only 38%, 44%, and 49%, respectively, whereas coincubation with IFN-γ reduced CE by only 18%, 27%, and 65%, respectively. In marked contrast to pHA-VLDL, both poly I and IFN-γ inhibited, by>95%, CE accumulation induced by copper-oxidized VLDL. These results demonstrate a novel mechanism for the conversion of type III VLDLs, their remnants, and EKO-VLDL into atherogenic particles and suggest that macrophage uptake of pHA-VLDL (1) requires catalytically active lipoprotein lipase, (2) involves acyl coenzyme A:cholesterol acyltransferase–mediated cholesterol esterification, and (3) involves pathways distinct from the SR-A.
Probucol Enhances Selective Uptake of HDL-Associated Cholesteryl Esters In Vitro by a Scavenger Receptor B-I–Dependent Mechanism
Authors: Rinninger Franz , Wang Nan , Ramakrishnan Rajasekhar , Jiang Xian Cheng , Tall Alan R.
Published: June 2011

Abstract
—Recently, the class B, type I scavenger receptor (SR-BI) has been shown to mediate the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CEs), ie, lipid uptake independent of HDL holoparticle uptake. In vivo, this selective uptake delivers CEs to the liver for excretion and to steroidogenic tissues for hormone synthesis. Probucol, a hydrophobic antioxidant drug, lowers plasma cholesterol in humans and rodents and may inhibit progression of atherosclerosis and postangioplasty restenosis. In this study, the effect of probucol on HDL selective CE uptake was investigated in mice and in cells expressing SR-BI. Probucol feeding lowered plasma HDL cholesterol and markedly increased selective CE uptake from HDL in the liver and adrenal glands. However, probucol did not alter SR-BI protein levels in membranes from these organs. When incubated with control Chinese hamster ovary (CHO) cells, HDL isolated from probucol-treated mice (P-HDL) and HDL from control mice (C-HDL) showed similar low selective uptake of CEs. However, when incubated with SR-BI–transfected CHO cells, P-HDL showed a 2-fold increase in selective uptake compared with C-HDL. In an adrenal cell line (Y1-BS1), which expresses SR-BI in an adrenocorticotropic hormone–inducible manner, P-HDL showed significantly greater selective CE uptake than did C-HDL, and the differential response was amplified by adrenocorticotropic hormone treatment. In contrast to P-HDL, incorporation of this compound into HDL in vitro did not result in stimulation of selective CE uptake by SR-BI–transfected CHO cells, even though a significant mass of probucol could be detected in the HDL preparation. The specific interaction of P-HDL with SR-BI in cell culture could be observed after only 24 hours of probucol feeding, when there were minimal changes in HDL size and composition. Thus, probucol or one of its metabolites increases selective CE uptake in vivo by modifying HDL in a way that causes enhanced interaction with SR-BI. The increased interaction of P-HDL with SR-BI in the liver and arterial wall may be partly responsible for the effects of probucol on atherosclerosis and restenosis.
Comparison of the Antithrombotic Effect of PEG-Hirudin and Heparin in a Human Ex Vivo Model of Arterial Thrombosis
Authors: Bossavy J. P. , Sakariassen K. S. , Rübsamen K. , Thalamas C. , Boneu B. , Cadroy Y.
Published: June 2011

Abstract
—Polyethylene glycol (PEG)-hirudin is a derivative of hirudin with a long plasma half-life. We have compared the efficacy of PEG-hirudin with unfractionated heparin (UH) in preventing arterial thrombosis. Arterial thrombus formation was induced ex vivo in 12 healthy human volunteers by exposing a tissue factor–coated coverslip positioned in a parallel-plate perfusion chamber to flowing nonanticoagulated human blood drawn directly from an antecubital vein at an arterial wall shear rate of 2600 s
−1
for 3.5 minutes. PEG-hirudin, UH, or saline (as control) were administered ex vivo through a heparin-coated mixing device positioned proximal to the perfusion chamber. Platelet and fibrin deposition was quantified by immunoenzymatic measure of the P-selectin and
d
-dimer content of dissolved plasmin-digested thrombi, respectively. UH was administered to a plasma concentration of 0.35 IU/mL. This concentration prolonged the activated partial thromboplastin time from 32±1 seconds to 79±4 seconds (
P
<0.01). UH did not significantly prevent platelet deposition. However, fibrin deposition was reduced by 39% (
P
<0.05). PEG-hirudin in plasma concentrations of 0.5, 2.5, and 5 μg/mL prolonged the activated partial thromboplastin time to 48±2, 87±4, and 118±4 seconds, respectively. In contrast to UH, PEG-hirudin prevented both platelet and fibrin deposition in a dose-dependent manner with a >80% reduction at 5 μg/mL (
P
<0.01). Furthermore, the plasma level of PEG-hirudin required to significantly prevent fibrin deposition (0.5 μg/mL) corresponded to a much shorter prolongation of activated partial thromboplastin time (48±2 seconds) than that needed for UH (79±4 seconds). Thus, our results are compatible with the view that thrombin is greatly involved in recruitment of platelets in evolving thrombi, and that PEG-hirudin is an effective agent for preventing arterial thrombosis in a human ex vivo experimental model.
Measuring Plasma Fibrinogen to Predict Stroke and Myocardial Infarction
Authors: Maresca Giulio , Di Blasio Anna , Marchioli Roberto , Di Minno Giovanni
Published: June 2011

Abstract
—Plasma fibrinogen is a major determinant of platelet aggregation and blood viscosity. The decrease in plasma fibrinogen by bezafibrate is associated with a decrease in the risk of reinfarctions. To strengthen the predictive value of plasma fibrinogen with respect to cardiovascular risk, we performed a meta-analysis of studies conducted between 1984 and 1998. Emphasis has been put on the relationship between high levels of plasma fibrinogen and fatal and/or nonfatal cardiovascular events in both the general population and in patients with previous cardiovascular events. Twenty-two studies (13 prospective, 5 cross-sectional, and 4 case-control) addressing the association between fibrinogen plasma concentrations and cardiovascular disease were analyzed. The overall estimate of risk of cardiovascular event in subjects with plasma fibrinogen levels in the higher tertile, was twice as high as that of subjects in the lower one (odds ratio, 1.99; 95% confidence interval, 1.85 to 2.13). High plasma fibrinogen levels were associated with an increased risk of cardiovascular disease in healthy as much as in high-risk individuals. A metaregression showed no confounding effects attributable to selected characteristics of retrieved studies. A subgroup analysis (study design, follow up, mean fibrinogen levels, percentage of smokers, and mean age) allowed us to conclude that fibrinogen is an independent risk factor for cardiovascular disease; that it interacts with major determinants of myocardial and cerebrovascular ischemia; and that, in secondary prevention studies, it enhances by 8% the prediction of future events by established risk factors. Thus, fibrinogen measurements should be encouraged to refine the overall risk profiles of individuals and to better tailor preventive interventions.