bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone Marrow Transplantation in Apolipoprotein E–Deficient Mice
Authors: Van Eck Miranda , Herijgers Nicole , Yates John , Pearce Nigel J. , Hoogerbrugge Peter M. , Groot Pieter H. E. , Van Berkel Theo J. C.
Published: June 2011

Abstract
Apolipoprotein E (apoE), a high-affinity ligand for lipoprotein receptors, is synthesized by the liver and extrahepatic tissues, including cells of the monocyte/macrophage lineage. Inactivation of the apoE gene in mice leads to a prominent increase in serum cholesterol and triglyceride levels and the development of premature atherosclerosis. In this study, the role of monocyte/macrophage-derived apoE in lipoprotein remnant metabolism and atherogenesis was assessed. The influence of apoE gene dosage on serum lipid concentrations was determined by transplantation of homozygous apoE-deficient (apoE
−/−
), heterozygous apoE-deficient (apoE
+/−
), and wild-type (apoE
+/+
) bone marrow in homozygous apoE-deficient mice. The concentration of apoE detected in serum was found to be gene dosage dependent, being 3.52±0.30%, 1.87±0.17%, and 0% of normal in transplanted mice receiving either apoE
+/+
, apoE
+/−
, or apoE
−/−
bone marrow, respectively. These low concentrations of apoE nevertheless dramatically reduced serum cholesterol levels owing to a reduction of VLDL and, to a lesser extent, LDL, while HDL levels were slightly raised. After 4 months on a “Western-type” diet, atherosclerosis was evidently reduced in mice transplanted with apoE
+/+
bone marrow, compared with control transplanted mice. To study the mechanism of the lipoprotein changes on bone marrow transplantation, the in vivo turnover of autologous serum (β)VLDL was studied. The serum half-life of (β)VLDL in transplanted mice, compared with control apoE-deficient mice, was shortened mainly as a consequence of an increased recognition and uptake by the liver. Analysis of the relative contribution of the liver parenchymal cells, endothelial cells, and Kupffer cells (liver tissue macrophages) indicated an increased uptake by parenchymal cells, while the relative contribution of Kupffer cells was decreased. In conclusion, macrophage-derived apoE can dose-dependently reduce hypercholesterolemia in apoE-deficient mice owing to increased recognition and uptake of (β)VLDL by parenchymal liver cells, leading to a decreased susceptibility to atherosclerosis.
Binding Properties and Inhibition of Platelet Aggregation by a Monoclonal Antibody to CD31 (PECAM-1)
Authors: Wu Xiao-Wei , Lian Eric C.-Y.
Published: June 2011

AbstractCD31/platelet endothelial cell adhesion molecule 1 (PECAM-1) is expressed on platelets, endothelial cells, myeloid cells, monocytes, and certain lymphocyte subsets. It has been shown that CD31 is involved in the homophilic and heterophilic cellular adhesion and leukocyte transendothelial migration, but little is known about the role of CD31 in platelet functions. Previously we have shown that monoclonal antibody (MAb) AAP2 produced in our laboratory bound to a 110-kD platelet antigen and gave an enhanced binding to activated platelet membrane. In this study we demonstrated that platelet lysate depleted of the antigen through adsorption by an AAP2-solidified affinity column was bound by MAbs against CD62 and CD42 but not by MAb 5.6E against CD31 or AAP2 on the immunoblot. Rabbit antibodies against CD31 completely inhibited the binding of AAP2 to platelets in the flow cytometry analysis. This indicates that AAP2 is specifically against CD31.125I-labeled AAP2 bound to resting platelets with 5587±1765 sites/platelet and aKdof 1 nmol/L and to thrombin-activated platelets with 17 625±4865 sites/platelet and aKdof 0.24 nmol/L. Addition of 10 μg/mL AAP2 inhibited the aggregation induced by 4 μmol/L ADP by 78.6%, 6 μmol/L epinephrine by 79.4%, 1 μg/mL collagen by 78.7%, and 0.25 U/mL thrombin by 29%. The platelet aggregation was completely restored when higher concentration of agonists were used. MAb 5.6E did not have any effect on platelet aggregation. These results suggest that AAP2 binds to a special epitope of CD31 on platelets and that CD31 is involved in platelet aggregation.
Antibodies Against Cardiolipin and Oxidatively Modified LDL in 50-Year-Old Men Predict Myocardial Infarction
Authors: Wu Ruihua , Nityanand Soniya , Berglund Lars , Lithell Hans , Holm Göran , Lefvert Ann Kari
Published: June 2011

Abstract
Autoantibodies against oxidatively modified low-density lipoproteins (oxLDL) and cardiolipin occur in patients with vascular diseases, including atherosclerosis. The ability of such antibodies to predict myocardial infarction (MI) was investigated in a prospective nested case-control study in which healthy 50-year-old men were followed up for 20 years. Raised levels of antibodies against oxLDL and cardiolipin at 50 years of age correlated positively with the incidence of MI and mortality related to MI 10 to 20 years later. IgG and IgA antibodies against cardiolipin were associated with MI between 50 to 60 years of age and IgG and IgA antibodies against oxLDL with MI at 60 to 70 years of age. Moreover, higher antibody levels were noted in those who died from acute MI in comparison to those who survived. The predictive power of IgA and IgG antibodies was strong and largely independent of that of other strong risk factors. In conclusion, raised levels of antibodies against oxLDL and cardiolipin may predict MI and MI-related death.
Plasma Levels of Nitrite/Nitrate and Platelet cGMP Levels Are Decreased in Patients With Atrial Fibrillation
Authors: Minamino Tetsuo , Kitakaze Masafumi , Sato Hiroshi , Asanuma Hiroshi , Funaya Hiroharu , Koretsune Yukihiro , Hori Masatsugu
Published: June 2011

Abstract
Patients with atrial fibrillation have been reported to exhibit abnormal hemostasis. Since nitric oxide (NO) exerts antithrombotic effects and attenuates platelet function, we evaluated two indicators of plasma NO levels, the plasma levels of nitrite and nitrate (NOx), and the levels of cGMP in platelets. We also examined whether indicators of plasma NO levels were associated with abnormalities in parameters related to platelet function, blood coagulation, and fibrinolysis. We evaluated 45 patients with chronic sustained atrial fibrillation (33 men and 12 women, age range 63±2 years) compared with 45 sex- and age- (±2 years) matched nonhospitalized subjects with sinus rhythm. There were no significant differences between the two groups in the incidence of risk factors for stroke except for ischemic heart disease or in echocardiographic parameters. Plasma levels of NOx measured using the Greiss reagent (mean [interquartile range]: 15.6 [9.5 to 25.7] versus 24.1 [14.2 to 40.8] μmol/L, n=45) and the platelet cGMP levels (0.33 [0.16 to 0.67] versus 0.63 [0.31 to 1.29] pmol/10
9
platelets, n=9) were significantly (
P
<.05) lower in the patients with atrial fibrillation than in the control subjects. Plasma levels of
d
-dimer, β-thromboglobulin, and fibrinogen were significantly (
P
<.05) higher in the patients with atrial fibrillation. The two groups did not differ as to the plasma levels of tissue plasminogen activator or plasminogen activator inhibitor-1. Our findings suggest that a decrease in plasma NO levels may account for the hemostatic abnormalities observed in patients with atrial fibrillation.
Evidence That Human Fcγ Receptor IIA (CD32) Subtypes Are Not Receptors for Oxidized LDL
Authors: Morganelli Peter M. , Groveman Debra S. , Pfeiffer Jason R.
Published: June 2011

AbstractSeveral lines of evidence suggest that clearance of oxidized LDL (oxLDL) immune complexes by macrophage IgG Fc receptors (FcγRs) plays a role in atherogenesis. OxLDL may also be cleared directly by FcγRs, as shown for murine FcγRII-B2. In humans, the homologous FcγR is FcγRIIA (CD32), which is abundantly expressed on monocytes and macrophages and shares 60% sequence identity with murine FcγRII-B2. As murine FcγRII-B2 and human FcγRIIA also share similar IgG ligand-binding properties, the purpose of this study was to test the hypothesis that human CD32 is a receptor for oxLDL. For these studies we used transfected Chinese hamster ovary (CHO) cells, monocytes, and cell lines that functionally express either of two FcγRIIA subtypes (R131 or H131) and assayed binding or degradation of several preparations of oxLDL. The integrity of all oxLDL preparations was checked by studying their ability to react with CHO cells expressing human type I scavenger receptors and by other characteristics of lipoprotein oxidation. Although we showed that each preparation of oxLDL could recognize class A or class B scavenger receptors, we did not detect any differences in the binding or degradation of any type of oxLDL preparation among control versus CHO cell transfectants. Using monocytes that express FcγRIIA and CD36, we showed that the binding of oxLDL was inhibited by antibodies to CD36, but not by FcγRIIA antibodies. Thus, the data do not support the hypothesis that human FcγRIIA is by itself a receptor for oxLDL. We conclude that human CD32 can mediate uptake of lipoprotein immune complexes, but does not mediate uptake of oxLDL in the absence of anti-oxLDL antibodies. OxLDL may interact with human mononuclear phagocytes directly via other types of receptors, such as class A and class B scavenger receptors or CD68.
Tetradecylthioacetic Acid Inhibits the Oxidative Modification of Low Density Lipoprotein and 8-Hydroxydeoxyguanosine Formation In Vitro
Authors: Muna Ziad A. , Doudin Khaled , Songstad Jon , Ulvik Rune J. , Berge Rolf K.
Published: June 2011

Abstract
Oxidative modification of low-density lipoprotein (LDL) is thought to play a key role in the formation of foam cells and in initiation and progression of atherosclerotic plaque. The hypolipidemic 3-thia fatty acids contain a sulfur atom and might therefore possess reducing (antioxidant) properties. Consequently, the effects of 3-thia fatty acids on the susceptibility of LDL particles to undergo oxidative modification in vitro were studied. Tetradecylthioacetic acid (TTA), incorporated into the LDL particle and increased the lag time of copper ion induced LDL oxidation in a dose-dependent manner. 80 μmol/L TTA reduced the generation of lipid peroxides during copper ion induced LDL oxidation (for 2 hours) by 100%, 2,2′-azobis-(2,4-dimethylvaleronitrile) induced LDL oxidation by 64%, and 2,2′-azobis-(2-amidinopropane hydrochloride) induced LDL oxidation (for 6 hours) by 21%. The electrophoretic mobility of the oxidized LDL was reduced by TTA in both copper ion and azo-compounds initiated oxidation. This fatty acid analogue was effectively able to reduce in a dose dependent manner the formation of 8-hydroxydeoxyguanosine from 2-deoxyguanosine with ascorbic acid as the radical producer. TTA bound copper(II) ions and did not reduce copper(II) to copper(I). It failed to scavenge the 1.1-diphenyl-2-picrylhydrazyl radicals. The results suggest that the modification of LDL in the lipid and protein moieties can be significantly reduced by TTA. This acid may exert its antioxidant effect partially through metal ion binding and through free radical scavenging.
Changes in Arterial Expression of Fibrinolytic System Proteins in Atherogenesis
Authors: Schneider David J. , Ricci Michael A. , Taatjes Douglas J. , Baumann Patricia Quinn , Reese Jeffrey C. , Leavitt Bruce J. , Absher P. Marlene , Sobel Burton E.
Published: June 2011

Abstract
Plasminogen activators (PAs) and their inhibitor, plasminogen activator inhibitor type-1 (PAI-1), have been implicated in modulation of luminal fibrinolysis and mural proteolysis contributing to atherogenesis.

Expression of PAs/PAI-1 (normalized to extracted tissue protein) was delineated by assays of conditioned media and of extracts from walls of human arterial segments in culture. Arterial specimens (n=39 from 26 subjects) were divided into four groups: normal (n=14), fatty streak (n=6), moderate atherosclerosis (mural thickening with <70% lumen obstruction, n=5), and severe atherosclerosis (mural thickening with >70% lumen obstruction, n=14). Paired samples from the same individual comprising a normal arterial segment and an atherosclerotic segment were evaluated also. A fourfold molar excess in PAI-1:t-PA was seen in conditioned media from samples with any evidence of atherosclerosis compared with normal specimens (normal 21±4, diseased 82±21,
P
≤.05). Compared with normal pairs, the tissue content of PAI-1 (ng) was increased in fatty streak lesions (n=3, normal 35±12, fatty streak 50±8,
P
≤.05); stable to decreased in moderate atherosclerosis (n=3, normal 34±3, moderate 22±7,
P
=.16); and increased in severe atherosclerosis (n=6, normal 48±9, severe 85±19,
P
≤.05). The tissue content of PAs (ng), though not increased in fatty streak lesions, was elevated in moderately and severely atherosclerotic segments (normal 0.7±0.2, moderate 1.6±0.1; normal 0.8±0.3, severe 2.1±0.3,
P
≤.05 for each comparison).

Atherogenesis is associated with decreased luminal fibrinolytic capacity that may exacerbate thrombosis. Decreased mural proteolysis in early atherogenesis may exacerbate matrix accumulation. Increased mural proteolysis later is associated with, and may potentiate, smooth muscle cell migration and proliferation.
αIIbβ3 Redistribution Triggered by Receptor Cross-Linking
Authors: Simmons Scott R. , Sims Paul A. , Albrecht Ralph M.
Published: June 2011

Abstract
Fibrinogen binding to αIIbβ3 on adherent, spread platelets triggers active, cytoskeletally-directed redistribution of fibrinogen/αIIbβ3 complexes on the platelet surface. Gold-conjugated fibrinogen, unlabeled, soluble fibrinogen, and individual fibrinogen molecules have been demonstrated to trigger receptor redistribution. Here we examine the respective roles of receptor cross-linking and ligand occupancy of receptors in initiating this movement. Monovalent, αIIbβ3-binding fibrinogen fragments RGDS and HHLGGAKQAGDV did not trigger receptor redistribution, suggesting that ligand binding to a single receptor is an insufficient stimulus. Binding of monoclonal antibodies 10E5, AP2, and AP3 to the receptor did not trigger receptor movement. However, cross-linking these receptor-bound monoclonal antibodies by polyclonal anti-mouse IgG or by conjugation of the anti-receptor antibody to large colloidal gold particles triggered receptor redistribution identical in rate, pattern, and final distribution to that previously seen with fibrinogen binding. We conclude that receptor cross-linking provides the signal for initiation of fibrinogen/αIIbβ3 complex redistribution on platelet surfaces.
Hemostatic Factors as Predictors of Ischemic Heart Disease and Stroke in the Edinburgh Artery Study
Authors: Smith F. B. , Lee A. J. , Fowkes F. G. R. , Price J. F. , Rumley A. , Lowe G. D. O.
Published: June 2011

Abstract
Plasma fibrinogen is a consistent predictor of ischemic heart disease (IHD) in prospective studies, but there are fewer data relating other hemostatic variables to IHD and also to stroke. We therefore studied the relationships of plasma fibrinogen, von Willebrand factor antigen, tissue plasminogen activator (TPA) antigen, factor VII, and fibrin D-dimer to incidence of IHD and stroke and determined whether any associations could be explained by conventional risk factors and baseline heart disease. In the Edinburgh Artery study, 1592 men and women aged 55 to 74 years, randomly sampled from the general population, were followed prospectively over 5 years to detect fatal and nonfatal IHD and stroke events. During the 5 years, 268 new vascular events were identified. Baseline plasma fibrinogen was independently related to risk of stroke in multivariate analysis that adjusted for cigarette smoking, LDL-cholesterol, systolic blood pressure, and preexisting IHD (relative risk [RR] 1.52, 95% confidence interval [CI] 1.17,1.98). TPA antigen, and fibrin D-dimer were also independently associated with risk of stroke (RR 1.69, 95% CI 1.22,2.35 and RR 1.96, 95% CI 1.12,3.41, respectively). Significant relationships were found between TPA antigen and myocardial infarction (
P
≤.05). In older men and women, increased coagulation activity and disturbed fibrinolysis are predictors of future vascular events (both IHD and stroke).
Soy Isoflavones Improve Systemic Arterial Compliance but Not Plasma Lipids in Menopausal and Perimenopausal Women
Authors: Nestel Paul J. , Yamashita Takeshi , Sasahara Takayuki , Pomeroy Sylvia , Dart Anthony , Komesaroff Paul , Owen Alice , Abbey Mavis
Published: June 2011

Abstract
The possibility that the heightened cardiovascular risk associated with the menopause, which is said to be ameliorated by soybeans, can be reduced with soy isoflavones was tested in 21 women. Although several were perimenopausal, all have been included. A placebo-controlled crossover trial tested the effects of 80-mg daily isoflavones (45 mg genistein) over 5- to 10-week periods. Systemic arterial compliance (arterial elasticity), which declined with age in this group, improved 26% (
P
<.001) compared with placebo. Arterial pressure and plasma lipids were unaffected. The vasodilatory capacity of the microcirculation was measured in nine women; high acetylcholine-mediated dilation in the forearm vasculature was similar with active and placebo treatments. LDL oxidizability measured in vitro was unchanged. Thus, one important measure of arterial health, systemic arterial compliance, was significantly improved in perimenopausal and menopausal women taking soy isoflavones to about the same extent as is achieved with conventional hormone replacement therapy.
Inhibition of Tissue Factor Gene Activation in Cultured Endothelial Cells by Curcumin
Authors: Pendurthi Usha R. , Williams J. Todd , Rao L. Vijaya Mohan
Published: June 2011

AbstractBinding of plasma factor VII(a) to tissue factor (TF) initiates the coagulation cascade. In health, TF is not expressed in endothelial cells. However, endothelial cells express TF in response to lipopolysaccharide (LPS), tumor necrosis factor-α (TNFα), and other biological stimuli. TF expression by endothelial cells is implicated in thrombotic disorders in patients with a variety of clinical disorders. In the present study, we demonstrate that curcumin (diferulolylmethane), a known anticarcinogenic and anti-inflammatory agent, inhibited phorbol 12-myristate 13-acetate (PMA), LPS, TNFα, and thrombin-induced TF activity and TF gene transcription in human endothelial cells. The present data show that curcumin prevented the activation of c-Rel/p65, which is essential for TF gene activation in endothelial cells, by impairing the proteolytic degradation inhibitor protein, IκBα. The data also show that curcumin downregulated AP-1 binding activity. The present studies are the first to demonstrate that PMA, but not LPS, TNFα, and thrombin, induced Egr-1 binding to the second serum-responsive region (SRR-2) of TF promoter and that curcumin inhibited the PMA-induced Egr-1 binding to SRR-2. Overall, the data suggest that the anticarcinogenic and anti-inflammatory properties of curcumin may be related to its ability to inhibit cellular gene expression regulated by transcription factors NF-κB, AP-1, and Egr-1.
Effect of α-Tocopherol (Vitamin E) and β-Carotene Supplementation on the Incidence of Intermittent Claudication in Male Smokers
Authors: Törnwall Markareetta E. , Virtamo Jarmo , Haukka Jari K. , Aro Antti , Albanes Demetrius , Edwards Brenda K. , Huttunen Jussi K.
Published: June 2011

Abstract
We examined the primary preventive effect of vitamin E (α-tocopherol) and β-carotene supplementation on intermittent claudication. The subjects—participants in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study—were male smokers aged 50 to 69 years who were randomly assigned to receive 50 mg of α-tocopherol daily, 20 mg of β-carotene daily, both, or placebo. At baseline, there were 26 289 men with no history or symptoms of intermittent claudication. The Rose questionnaire on intermittent claudication was administered annually to discover incident cases. We observed 2704 cases of first occurrence of typical intermittent claudication during a median follow-up time of 4.0 years. Compared with placebo, the adjusted relative risk for typical intermittent claudication among those who received α-tocopherol only was 1.11 (95% confidence interval, 1.00-1.24); among those who received α-tocopherol and β-carotene, 1.02 (0.91-1.13); and among those who received β-carotene only, 1.02 (0.92-1.14). When we compared the α-tocopherol–supplemented subjects with those who received no α-tocopherol, the adjusted relative risk for typical intermittent claudication was 1.05 (0.98-1.14), and for β-carotene–supplemented subjects compared with those who did not receive β-carotene, the relative risk was 0.96 (0.89-1.04). In conclusion, no primary preventive effect on intermittent claudication was observed among middle-aged male smokers who were supplemented with α-tocopherol, β-carotene, or both.
Cholesterol Reduction and Clinical Benefit
Authors: Fager Gunnar , Wiklund Olov
Published: June 2011

Abstract
Encouraging intervention trials drive our expectations toward more aggressive cholesterol-lowering therapies, lower target levels, and less severe hypercholesterolemia. Available studies may predict which patients, degrees of total cholesterol (TC) reduction, and baseline and target levels of TC provide the most clinical benefit. Data were pooled from seven primary and nine secondary controlled trials with major coronary heart disease (CHD) events as primary endpoints. The analysis showed that we can expect large reductions in CHD from TC reduction in primary and secondary prevention. However, the reduction is much larger in subjects with high TC and/or previous CHD events. The percent reduction in CHD increased exponentially with increasing percent TC reductions, which predicted >70% of the change in CHD. Consequently, we cannot expect cost-effective clinical benefits from mean reductions in TC >15 (LDL cholesterol >20)%. The TC level at the study endpoint correlated with CHD incidence irrespective of the study group and explained almost 45% of CHD incidence. The relationship was progressive and leveled off at a TC level below about 150 mg/dL (3.9 mmol/L) (LDL cholesterol ≈110 mg/dL [≈2.8 mmol/L]). Little extra clinical benefit can be expected from further reductions. We can expect an average 2% reduction in CHD events per percent reduction in TC. We can also expect a 2-fold greater clinical benefit among subjects with high initial TC levels than among those with low levels. Finally, we can expect that the cholesterol-attributable risk is reset to that predicted by the TC level achieved within 4 to 6 years.
Peripheral Vascular Stenosis in Apolipoprotein E-Deficient Mice
Authors: Seo Hong Seog , Lombardi Donna M. , Polinsky Patti , Powell-Braxton Lyn , Bunting Stuart , Schwartz Stephen M. , Rosenfeld Michael E.
Published: June 2011

AbstractA systematic analysis of the distribution of advanced atherosclerotic lesions was undertaken in chow-fed, 9-month-old apolipoprotein (apo) E-deficient mice to identify sites amenable for study of mechanisms of formation of stenotic lesions. The arterial tree was dissected intact and included medium-sized arteries in the extremities as well as arteries of the head and neck. The most reproducible lesions were seen in the ascending aorta and in the carotid, femoral, and popliteal arteries. Casting of the vascular tree provided additional verification of the presence of lumen narrowing in the external branches of the carotid artery. Consistent with what has been observed in human atherosclerotic arteries, there was dilation in response to lesion growth and no correlation between lesion mass and lumen loss in the mouse arteries. This adaptation was especially true in the ascending aorta, where normal lumen size was maintained at atherosclerotic sites. In contrast, the external carotid arteries were stenotic in 9 of 12 animals. Here too, however, loss of lumen did not correlate with lesion mass but did correlate with adventitial inflammation and medial atrophy. Lumen narrowing also occurred most frequently at sites where extracellular cholesterol clefts were a prominent part of the lesion. These data suggest that the stenotic process in advanced atherosclerotic vessels may depend on death of medial smooth muscle cells, possibly in response to inflammatory changes in the plaque or adventitia.
A Prospective Case-Control Study of Lipoprotein(a) Levels and Apo(a) Size and Risk of Coronary Heart Disease in Stanford Five-City Project Participants
Authors: Wild Sarah H. , Fortmann Stephen P. , Marcovina Santica M.
Published: June 2011

Lipoprotein(a) [Lp(a)] is formed by the assembly of LDL particles and a carbohydrate-rich protein, apolipoprotein(a) [apo(a)], which has a high degree of structural homology with plasminogen. While the majority of retrospective studies have found an association between Lp(a) level and cardiovascular disease (CVD), the few prospective studies to date have reported contradictory results. We conducted a nested case-control study using the participants in the Stanford Five-City Project, a long-term CVD prevention trial. Participants with an incident possible or definite myocardial infarction or coronary death were matched to a single control subject for age, sex, ethnicity, residence in a treatment or control city, and time of survey. This process yielded 134 case-control pairs, 90 male and 44 female, for whom plasma was available for analysis of Lp(a). Lp(a) values in nanomoles per liter were determined by an enzyme-linked immunoassay that measures Lp(a) independently of apo(a) size polymorphism. Apo(a) size isoforms were determined by SDS–agarose gel electrophoresis. Median Lp(a) level in male cases was almost double that in control subjects (41.8 versus 21.2 nmol/L;
P
<.01); in female cases, median Lp(a) was 34% higher than in control subjects (32.5 versus 21.2 nmol/L), but this difference was not statistically significant. Among the male cases, there was an increased frequency of small apo(a) isoforms, while no significant difference was found in apo(a) size between female cases and control subjects. The association between Lp(a) level and case-control status in men was independent of total, HDL, and non-HDL cholesterol levels, as well as apo(a) size isoform, cigarette smoking, blood pressure, and obesity. In men, the most efficient threshold value of Lp(a) concentration for separating cases and control subjects was 35 nmol/L; the odds ratio for being a case above this level compared with below was 2.84 (95% confidence interval: 1.53-5.27,
P
<.001). This study provides strong evidence that Lp(a) level is a prospective, independent risk factor for developing coronary artery disease in men and indicates that the size of apo(a) may also play a role. The lack of a significant association in women deserves further evaluation in larger studies.
Polymorphisms of Factor V, Factor VII, and Fibrinogen Genes
Authors: Wang X.L. , Wang J. , McCredie R.M. , Wilcken D.E.L.
Published: June 2011

We explored the associations between G→A mutations of factor V and factor VII genes and the
Hae
III polymorphism of the fibrinogen gene and the severity of coronary artery disease (CAD), as assessed angiographically in 545 white Australian patients (388 male and 157 female) aged ≤65 years. We also assessed the relations with other potentially atherogenic variables. Elevated fibrinogen levels were associated with more severe CAD (
P
<.05), but none of the factor V, factor VII, and fibrinogen DNA variants were predictive of CAD severity, as assessed by the number of significantly diseased vessels (>50% luminal obstruction). The rare allele frequencies of factor V (
A
allele), factor VII (
M2
allele), and fibrinogen (
H2
allele) were .025, .114, and .201 for men and .022, .077, and .169 for women, respectively, and were not different from those in healthy whites. In the patient population, there was a strong, positive association between lifetime smoking dose (in pack-years) and circulating fibrinogen levels (
r
=.184,
P
=.001). This association was stronger than that between current smoking habit and fibrinogen and is consistent with a dosage effect. However, there was no significant contribution of fibrinogen genotype to fibrinogen levels in this patient population. We conclude that elevated fibrinogen levels are associated not only with the occurrence of CAD but also with more severe CAD and that measurement of DNA variants of the factor V, factor VII, and fibrinogen genes that we assessed may not provide information in predicting CAD severity in addition to that obtained by measuring circulating levels of the relevant clotting factors. There is, moreover, a positive dosage effect (in pack-years) of smoking on circulating fibrinogen levels.
Differential Roles of AT
1
and AT
2
Receptor Subtypes in Vascular Trophic and Phenotypic Changes in Response to Stimulation With Angiotensin II
Authors: Sabri Abdelkarim , Levy Bernard I. , Poitevin Pierre , Caputo Lidia , Faggin Elisabetta , Marotte Françoise , Rappaport Lydie , Samuel Jane Lyse
Published: June 2011

The aim of this study was to investigate the roles of angiotensin II (Ang II) receptor subtypes 1 (AT
1
) and 2 (AT
2
) in producing vascular wall hypertrophy and qualitative changes in smooth muscle cell gene expression. Wistar rats were treated for 23 days with osmotic minipumps containing solvent and either Ang II (120 ng·kg
−1
·min
−1
) or
PD123319
(30 mg·kg
−1
·d
−1
), an AT
2
receptor antagonist. In addition, rats receiving solvent and either Ang II or
PD123319
were given losartan, an AT
1
receptor antagonist, in the drinking water (10 mg·kg
−1
·d
−1
). Vascular wall hypertrophy and smooth muscle phenotype were characterized by morphometric analysis combined with immunohistochemistry. Ang II–induced hypertension was associated with the development of medial hypertrophy of the aorta and coronary arteries accompanied by reversion of vascular smooth muscle cells (VSMCs) toward an immature phenotype, as shown by the expression of cellular fibronectin and nonmuscle myosin. Losartan treatment, which restored normal arterial pressure, prevented all these changes.
PD123319
treatment, which had no effect on blood pressure, prevented only vascular hypertrophy, with no effect on VSMC phenotype. Administration of only losartan to normal rats reproduced the Ang II–induced vascular hypertrophy, with no effect on VSMC phenotype. Taken together, these results suggest that (1) the trophic effect of Ang II on VSMCs is mediated via AT
2
receptor subtypes and (2) changes in VSMC phenotypes are triggered mainly through AT
1
receptor subtypes.
A Role for Tyrosine Phosphorylation in Generation of Inositol Phosphates and Prostacyclin Production in Endothelial Cells
Authors: Helgadóttir Anna , Halldórsson Haraldur , Magnúsdóttir Kristín , Kjeld Matthías , Thorgeirsson Gudmundur
Published: June 2011

We have examined the effects of the protein tyrosine phosphatase inhibitor pervanadate on activation of signal transduction in human umbilical vein endothelial cells. Endothelial cells responded to pervanadate treatment by increasing tyrosine phosphorylation of cellular proteins, including phospholipase C (PLC)
γ1
, generating inositol phosphates (IPs), releasing arachidonic acid, and producing prostacyclin (prostaglandin [PG] I
2
). The dose and time responses for these events were similar. Tyrosine phosphorylation and formation of IPs in response to pervanadate were reduced by both staurosporine and genistein. Short-term incubation with the phorbol ester 12-
O
-tetradecanoylphorbol 13-acetate, which inhibits thrombin-induced IP generation, did not affect the IP response to pervanadate. To investigate the possible involvement of tyrosine phosphorylation in thrombin or histamine-induced IP generation and PGI
2
production, we examined the effects of costimulation with pervanadate and either thrombin or histamine. These responses proved to be different. While the tyrosine phosphorylation of PLC
γ1
was enhanced after cotreatment with thrombin and pervanadate compared with pervanadate alone, costimulation with pervanadate and histamine resulted in no more tyrosine phosphorylation of PLC
γ1
than after pervanadate alone. Similarly, while cotreatment with pervanadate and thrombin caused synergistic increase in IP generation, costimulation with pervanadate and histamine resulted in an additive response. However, PGI
2
responses to costimulation of pervanadate with either thrombin or histamine were both synergistic. Furthermore, stimulation with histamine, thrombin, or pervanadate all caused tyrosine phosphorylation of a mitogen-activated protein kinase (ERK1/p44). The results suggest that a tyrosine phosphorylation–dependent mechanism has a role in the phosphoinositide signal transduction pathway of human endothelial cells. Moreover, thrombin- but not histamine-induced generation of IPs appears to be partly caused by tyrosine phosphorylation of PLC
γ1
.
Involvement of Phosphatidylcholine Hydrolysis by Phospholipase D in Extracellular ATP-Induced Arachidonic Acid Release in Aortic Smooth Muscle Cells
Authors: Shinoda Junji , Suzuki Atsushi , Oiso Yutaka , Kozawa Osamu
Published: June 2011

We investigated the effect of extracellular ATP on phosphatidylcholine-hydrolyzing phospholipase D activity and the role of phospholipase D activation in extracellular ATP-induced arachidonic acid release in cultured rat aortic smooth muscle cells. ATP significantly stimulated the formation of choline in a dose-dependent manner in the range between 0.01 and 0.5 mmol/L. However, ATP had no effect on the formation of phosphocholine. Staurosporine, an inhibitor of protein kinases, did not affect the ATP-induced formation of choline. ATP significantly stimulated arachidonic acid release in a dose-dependent manner in the range between 0.01 and 0.5 mmol/L.
dl
-Propranolol hydrochloride (propranolol), an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the ATP-induced release of arachidonic acid. 1,6-Bis(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a potent and selective inhibitor of diacylglycerol lipase, reduced ATP-induced arachidonic acid release. Quinacrine, a phospholipase A
2
inhibitor, suppressed ATP-induced arachidonic acid release. Both propranolol and RHC-80267 markedly inhibited the ATP-induced synthesis of 6-ketoprostaglandin F
1α
, a stable metabolite of prostacyclin. These results strongly suggest that extracellular ATP activates phosphatidylcholine-hydrolyzing phospholipase D independently of protein kinase C in aortic smooth muscle cells and that the arachidonic acid release induced by extracellular ATP is mediated, at least in part, through phosphatidylcholine hydrolysis by phospholipase D activation.
Localization of Nonpancreatic Secretory Phospholipase A
2
in Normal and Atherosclerotic Arteries
Authors: Hurt-Camejo Eva , Andersen Sonja , Standal Rune , Rosengren Birgitta , Sartipy Peter , Stadberg Elizabeth , Johansen Berit
Published: June 2011

Secretory nonpancreatic type II phospholipase A
2
(snpPLA
2
) hydrolyzes fatty acids at the sn-2 position in phospholipids releasing free fatty acids (FFAs) and lysophospholipids. These products may act as intracellular second messengers or can be further metabolized into proinflammatory lipid mediators. The presence of snpPLA
2
in extracellular fluids and serum during inflammation has suggested a role of the enzyme in this process. However, the presence of snpPLA
2
in a variety of normal tissues suggests that snpPLA
2
may also have physiological functions. Atherosclerosis appears to have an inflammatory component. Here we report on the snpPLA
2
localization in normal and atherosclerotic lesions and on the properties of the isolated enzyme. A strong snpPLA
2
immunoreactivity was observed in the arterial media that was colocalized with α-actin–positive vascular smooth muscle cells (SMCs) in both normal and atherosclerotic vessels. In aortic atherosclerotic lesions, snpPLA
2
was observed colocalized with CD68-positive macrophages and HHF-35–positive SMCs and extracellularly in the lipid core. snpPLA
2
was isolated from human normal arteries and from aorta with lesions. The enzyme was isolated by acid extraction of normal arterial tissues followed by immunoaffinity chromatography. The purified snpPLA
2
had an expected molecular weight of 14 kD by polyacrylamide gel electrophoresis and appeared as a single band in immunoblotting. The enzymatic activity was followed by measuring release of fatty acids from phospholipid liposomes or LDL as substrates. The enzymatic activity was inhibited with two specific inhibitors for human snpPLA
2
: (1) monoclonal antibody 187 and (2) LY311727, a synthetic selective inhibitor. The mRNA for snpPLA
2
was detected with reverse transcriptase polymerase chain reaction. These results indicate that snpPLA
2
is present in human arteries and that it is able to hydrolyze phospholipids in LDL. The results support the hypothesis that snpPLA
2
can release proinflammatory lipids at places of LDL deposition in the arterial wall.
P-Selectin and Vascular Cell Adhesion Molecule-1 Are Focally Expressed in Aortas of Hypercholesterolemic Rabbits Before Intimal Accumulation of Macrophages and T Lymphocytes
Authors: Sakai Atsushi , Kume Noriaki , Nishi Eiichiro , Tanoue Kenjiro , Miyasaka Masayuki , Kita Toru
Published: June 2011

The accumulation of monocyte/macrophages and T lymphocytes in arterial intima is a hallmark of early atherogenesis. To investigate the temporal relationships between endothelial expression of adhesion molecules (eg, P-selectin and vascular cell adhesion molecule-1 [VCAM-1]) and intimal accumulation of macrophages and T lymphocytes, immunostaining was performed by using serial frozen sections from intercostal branch points of thoracic aortas of New Zealand White rabbits that had been fed a 0.3% cholesterol diet. After 1 week of cholesterol feeding, neither macrophages nor T lymphocytes were detected, although endothelial expression of P-selectin and VCAM-1 was observed. After 3 weeks, macrophages were detectable in 75% and T lymphocytes were present in 25% of the rabbits. Expression of P-selectin and VCAM-1 was sustained until 10 weeks. Infiltration of T lymphocytes was restricted in areas in which macrophages were accumulated and did not appear to precede macrophage infiltration. E-selectin expression was not detectable before accumulation of mononuclear leukocytes; however, very few endothelial cells covering foam cell lesions expressed E-selectin after 6 weeks. Similar results were obtained in Watanabe heritable hyperlipidemic rabbits aged 1, 2, and 3 months. Taken together, localized expression of P-selectin and VCAM-1 may play a key role in the initial recruitment of macrophages and T lymphocytes in early atherogenesis.
Reduced Responsiveness of Hypercholesterolemic Rabbit Aortic Smooth Muscle Cells to Nitric Oxide
Authors: Weisbrod Robert M. , Griswold Mark C. , Du Yue , Bolotina Victoria M. , Cohen Richard A.
Published: June 2011

The response to nitric oxide of intracellular free Ca
2+
levels, measured by fura 2 fluorimetry, and cyclic GMP, measured by RIA, was evaluated on smooth muscle cells of the thoracic aorta in primary culture from normal and cholesterol-fed rabbits. Relaxation to acetylcholine and nitric oxide was also determined in isolated rings of aorta. After 10 weeks of high-cholesterol diet, the intact aorta relaxed less to both acetylcholine and nitric oxide. In cultured cells from hypercholesterolemic rabbits, intracellular Ca
2+
oscillated, and the mean Ca
2+
levels were approximately twofold greater than in normal aortic cells. Nitric oxide failed to affect basal Ca
2+
in either cell type. The peak and sustained rise in intracellular Ca
2+
induced by angiotensin II (10
−7
mol/L) were similar in the two cell types. However, nitric oxide (10
−10
to 10
−6
mol/L) decreased the sustained Ca
2+
levels to a significantly smaller extent in cells from cholesterol-fed rabbits. In addition, in cells from hypercholesterolemic rabbits, nitric oxide added before angiotensin II inhibited to a smaller degree the transient increase in intracellular free Ca
2+
caused by angiotensin II in the nominal absence of extracellular Ca
2+
, as well as the increase in Ca
2+
associated with the addition of extracellular Ca
2+
. Measurements of fura 2 quenching caused by Mn
2+
influx confirmed that nitric oxide inhibited the entry of extracellular divalent cations significantly less in cells from hypercholesterolemic rabbits. Basal levels of cyclic GMP were significantly less than normal, and nitric oxide increased levels of cyclic GMP to a significantly smaller degree in cells from cholesterol-fed rabbits. These data indicate a substantial resistance to nitric oxide action in aortic smooth muscle cells of cholesterol-fed rabbits. This observation is consistent with the notion that resistance of smooth muscle cells to nitric oxide contributes to abnormal endothelium-dependent vasodilatation during hypercholesterolemia and can play a role in the pathogenesis of atherosclerosis.
Uptake by J774 Macrophages of Very-Low-Density Lipoproteins Isolated From ApoE-Deficient Mice Is Mediated by a Distinct Receptor and Stimulated by Lipoprotein Lipase
Authors: Hendriks Wendy L. , van der Sman-de Beer Femke , van Vlijmen Bart J.M. , van Vark Leonie C. , Hofker Marten H. , Havekes Louis M.
Published: June 2011

Abstract
Apolipoprotein (apo) E–deficient mice display marked accumulation in the plasma of VLDL deficient in both apoE and apoB100 but containing apoB48, apoA-I, apoCs, and apoA-IV. Since apoE-deficient mice develop severe atherosclerotic lesions with lipid-laden macrophages, we reasoned that the uptake of lipoproteins by intimal macrophages can take place in the absence of both apoE and apoB100. To get more insight into the mechanism of foam cell formation in apoE-deficient mice, we measured the interaction of VLDL from apoE-deficient mice (apoE
null
VLDL) with the murine macrophage cell line J774. Scatchard analysis revealed that apoE
null
VLDL is bound to J774 cells with a
K
d
value comparable to that of control VLDL (8.1 versus 4.7 μg/mL) and with a B
max
value about half that of control VLDL (40 versus 70 ng/mg cell protein, respectively). ApoE
null
VLDL is also taken up and degraded by J774 macrophages via a high-affinity process less efficiently than control mouse VLDL (6-fold and 50-fold less efficiently, respectively). In line with this observation, incubation of J774 cells with 50 μg/mL apoE
null
VLDL for 24 hours resulted in an increase in intracellular cholesteryl ester (CE) content, although 5-fold less pronounced than after incubation with 50 μg/mL control mouse VLDL. Under the conditions applied, simultaneous addition of 5 μg/mL lipoprotein lipase (LPL) stimulated the cellular uptake and degradation of apoE
null
VLDL about 10-fold and resulted in a 5-fold stimulation of the intracellular CE accumulation, from 9±2 to 46±5 μg CE per milligram cell protein. In contrast to control mouse VLDL, apoE
null
VLDL could not compete with
125
I-labeled LDL for binding to the LDL receptor of J774 cells. Furthermore, neither LDL nor acetylated LDL could compete with
125
I-labeled apoE
null
VLDL for binding to these cells, whereas control mouse VLDL, VLDL from a hypertriglyceridemic patient, and apoE
null
VLDL itself were efficient competitors. Thus, VLDL from apoE-deficient mice is taken up by J774 macrophages through recognition by a distinct receptor, which could be the triglyceride-rich lipoprotein receptor. We conclude that in apoE-deficient mice, foam cell formation occurs via a receptor-mediated uptake of apoE
null
VLDL, which can be stimulated by the presence of LPL.
7E3 Monoclonal Antibody Directed Against the Platelet Glycoprotein IIb/IIIa Cross-reacts With the Leukocyte Integrin Mac-1 and Blocks Adhesion to Fibrinogen and ICAM-1
Authors: Simon Daniel I. , Xu Hui , Ortlepp Susan , Rogers Campbell , Rao Navaneetha K.
Published: June 2011

AbstractRecent clinical trials suggest that blockade of integrins is a promising strategy for the treatment of acute coronary syndromes. Administration of 7E3 monoclonal antibody (mAb) Fab fragment (c7E3 Fab) directed against platelet integrin IIb/IIIa (αIIbβ3, CD41/CD61) reduces acute ischemic complications of coronary angioplasty and clinical restenosis at 6 months. However, 7E3 mAb is not selective for platelet IIb/IIIa but also cross-reacts with the leukocyte integrin Mac-1 (αMβ2, CD11b/CD18) and the vitronectin receptor (αvβ3, CD51/CD61). Information regarding how this mAb may affect other cells important in vascular repair is scant. Potential interactions of c7E3 Fab with inflammatory (ie, monocytes and neutrophils), vascular smooth muscle, and endothelial cells may contribute to the in vivo actions of c7E3 Fab. In this study we explored the binding of 7E3 to monocytic cells and the functional effect of 7E3 and c7E3 Fab on Mac-1–mediated adhesion to fibrinogen (FGN) and intercellular adhesion molecule-1 (ICAM-1), ligands abundant in the injured vessel wall. Flow cytometry demonstrated that 7E3 bound to THP-1 monocytic cells and identified a subpopulation (≈10%) of Mac-1 that was qualitatively similar to that recognized by CBRM1/5, a mAb directed to an activation-specific neoepitope present on a subset of Mac-1 molecules. mAb 7E3 bound to K562 cells transfected with just the α subunit (CD11b) of Mac-1 but not to nontransfected cells, confirming a direct interaction between 7E3 and Mac-1. mAb 7E3 and c7E3 Fab blocked the adhesion of Mac-1–bearing cells to FGN (80±11% and 78±9% inhibition, respectively) and ICAM-1 (62±14% and 62±17%). Both 7E3 and c7E3 Fab significantly inhibited (70±6% and 62±26%) soluble FGN binding to human peripheral blood monocytes. Thus, c7E3 Fab cross-reacts with the CD11b subunit of Mac-1 and interrupts cell-extracellular matrix and cell-cell adhesive interactions and may thereby influence the recruitment of circulating monocytes to sites of vessel injury. Given the recent evidence that adherent and infiltrating monocyte number directly correlates with the extent of neointimal hyperplasia, inhibition of Mac-1–dependent adhesion and IIb/IIIa-dependent function by c7E3 Fab may jointly contribute to the regulation of vascular repair and to the sustained clinical benefits observed with c7E3 Fab after angioplasty.