bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake of vWF–Anti-vWF Complexes by Platelets in Suspension
Authors: White J.G. , Krumwiede M.D. , Cocking-Johnson D.J. , Escolar G.
Published: June 2011

Efforts to identify the translocation of glycoprotein (GP) Ib/IX receptors, either bound to von Willebrand factor (vWF) or not, from exposed surfaces to interior membranes of thrombin-activated platelets in suspension have been unsuccessful. To observe vWF uptake by platelets, we added an anti-vWF antibody and staphylococcal protein A–gold (to act as a marker for the antibody) to an incubation medium containing washed platelets and bovine plasma vWF or ristocetin-activated human vWF. Thin sections of platelets incubated for 10, 20, or 30 minutes with vWF but without antibody revealed no internalization and minimal changes in the original discoid form. Over the same 30-minute period with anti-vWF, however, GPIb/IX-vWF–anti-vWF complexes were cleared from cell exteriors to channels of the open canalicular system. Engorgement of the open canalicular system with vWF multimers resulted in changes in shape, internal transformation, and degranulation. Physical changes associated with anti-vWF–induced uptake of vWF are not seen in platelets that are involved in hemostatic plug formation or clot retraction.
Selective Recruitment of ApoB-48 for the Assembly of VLDL in Rat Triacylglycerol-Enriched Hepatocytes
Authors: Coussons Peter J. , Bourgeois Catherine S. , Wiggins David , Gibbons Geoffrey F.
Published: June 2011

Primary rat hepatocyte cultures were enriched in cellular triacylglycerol (TAG) by exposure to extracellular oleate for 3 days. Control cells were cultured for the same time without oleate. The large increase in TAG secretion into the medium of TAG-enriched cells during the final 24 hours (225±30 versus 40±10 μg/mg cell protein [control cells],
P
<.01) was not accompanied by a similar change in apolipoprotein B (apoB) secretion (4.22±0.94 versus 3.72±0.75 μg/mg per 24 hours, respectively). Instead, TAG-enriched cells recruited a larger proportion of apoB for the synthesis of very low density lipoprotein (VLDL), the secretion of which was substantially higher under these circumstances (1.46±0.39 versus 0.34±0.06 μg apoB per milligram cell protein per 24 hours,
P
<.05). The increase in VLDL assembly was accompanied by a selective 2.5-fold increase (
P
<.05) in the specific recruitment of apoB-48. There was no significant increase in the amount of apoB-100, which appeared in the VLDL fraction when cells were enriched with TAG. Under these circumstances there was an increase in net cellular synthesis of apoB-48

(5524±667 versus 2505±598 disintegrations per minute per milligram protein per hour,
P
<.05). The net cellular synthesis of apoB-100 was unchanged compared with that observed in control cell cultures (1548±237 versus 2000±897 dpm/mg per hour, respectively). A large proportion of the total secreted apoB was associated with small particles of density higher than VLDL, even when VLDL output was maximally stimulated, suggesting that apoB was oversecreted and in excess of the cells' requirement to transport TAG.
Macrophages Stimulate Cholesteryl Ester Accumulation in Cocultured Smooth Muscle Cells Incubated With Lipoprotein-Proteoglycan Complex
Authors: Vijayagopal Parakat , Glancy D. Luke
Published: June 2011

Foam cells of atherosclerotic lesions originate from both macrophages and smooth muscle cells (SMCs). We explored the mechanism by which SMCs may become lipid laden. Confluent bovine aortic SMCs were cocultured with P388D
1
macrophages, and the cocultures were incubated for various times with low-density lipoprotein (LDL), acetyl-LDL, or lipoprotein-proteoglycan (PG) complex isolated from human atherosclerotic lesions. Macrophages were then removed from the SMCs and the cholesteryl ester (CE) content of the SMCs was quantitated. Lipoprotein-PG complex but not LDL or acetyl-LDL produced a 6-fold to 9-fold stimulation of CE synthesis and a 4.4-fold increase in cellular CE mass in cocultured SMCs relative to control SMCs. In similar studies with human aortic SMC-macrophage cocultures, macrophages stimulated lipoprotein-PG complex–mediated CE synthesis 7-fold to 13-fold and CE mass 7.8-fold in cocultured SMCs compared with SMCs cultured alone. CE synthesis that was mediated by lipoprotein-PG complex was dose dependent and increased linearly with time. Incubation of lipoprotein-PG complex with SMC-macrophage cocultures but not with SMCs or macrophages alone resulted in aggregation of the complex and stimulation of cholesterol esterification in SMCs by the conditioned media containing the aggregated complex. Cytochalasin D, an inhibitor of phagocytosis, inhibited CE synthesis mediated by lipoprotein-PG complex by 73%, whereas polyinosinic acid, an inhibitor of the scavenger receptor, had no effect. Upregulation or downregulation of apolipoprotein B,E receptors did not affect the lipoprotein-PG complex–mediated CE synthesis by cocultured SMCs. Lipoprotein-PG complex did not stimulate CE synthesis in SMCs cocultured with aortic endothelial cells or macrophages cocultured with SMCs. These results indicate that macrophages can stimulate CE synthesis and accumulation in cocultured SMCs when incubated with lipoprotein-PG complexes isolated from atherosclerotic lesions. This could be a potential mechanism for myocyte foam cell formation.
Arterial Smooth Muscle Cell Heparan Sulfate Proteoglycans Accelerate Thrombin Inhibition by Heparin Cofactor II
Authors: Shirk Rebecca A. , Church Frank C. , Wagner William D.
Published: June 2011

Heparin cofactor II (HCII) is a potent thrombin inhibitor in the presence of heparin and dermatan sulfate, glycosaminoglycans that accelerate the inhibition reaction. HCII is postulated to be an extravascular thrombin inhibitor that is stimulated physiologically by dermatan sulfate proteoglycans. To understand how thrombin activity may be downregulated within the artery wall, cultured monkey aorta smooth muscle cell (SMC) proteoglycans were tested for their ability to accelerate thrombin inhibition by HCII. Early confluent SMC monolayers increased thrombin-HCII inhibition rates 2-fold to 4-fold compared with reactions in cell-free control wells (7.3±0.5 versus 2.7±0.2×10
4
mol·L
−1
·min
−1
, with and without SMCs, respectively; n=7 experiments). Extracellular matrix obtained by cell monolayer removal also accelerated the thrombin-HCII inhibition reaction 3-fold to 5-fold. Rate increases were abolished by Polybrene or protamine sulfate. Pretreatment of monolayers with heparitinase I (and of extracellular matrix with HNO
2
) to degrade heparan sulfate blocked the thrombin-HCII inhibition rate increase. In contrast, pretreatment with chondroitinase ABC in the presence of proteinase inhibitors had no effect. “Pericellular” (cell surface– and extracellular matrix–derived) SMC heparan sulfate proteoglycans (HSPGs) were purified and fractionated by charge on DEAE-Sephacel. At a concentration of 1 μg/mL hexuronic acid, high-charge HSPG stimulated a 7-fold thrombin-HCII inhibition rate increase relative to reactions without proteoglycan, whereas low-charge HSPG induced a 2-fold rate increase. In comparison, an 18-fold rate increase was observed with 1 μg/mL dermatan sulfate proteoglycan purified from SMC culture media. These results indicate that SMC HSPG could contribute significantly to thrombin inhibition by HCII in the artery wall.
Correlates of Thrombin Markers in an Elderly Cohort Free of Clinical Cardiovascular Disease
Authors: Cushman Mary , Psaty Bruce M. , Macy Elizabeth , Bovill Edwin G. , Cornell Elaine S. , Kuller Lewis H. , Tracy Russell P.
Published: June 2011

Studies suggest that thrombosis is important in the progression of atherosclerotic lesions. The biochemical markers prothrombin fragment 1-2 and fibrinopeptide A reflect in vivo thrombin generation and activity, respectively. As such, they are markers that might be associated with cardiovascular risk. From the Cardiovascular Health Study, a cohort study of 5201 persons over 65 years of age, 399 persons free of clinical cardiovascular disease (CVD) at the baseline examination were selected for study of specialized markers of hemostasis. We report the cross-sectional relationships of the thrombin markers to CVD risk factors and measures of subclinical CVD. The range of fragment 1-2 was 0.12 to 0.85 nmol/L. The range of fibrinopeptide A was 0.9 to 44.1 μg/L. High levels of fragment 1-2 and fibrinopeptide A were associated with age, with levels higher in women than men. Fragment 1-2 was associated with smoking; high levels of triglyceride, creatinine, and C-reactive protein; and low levels of glucose. Fibrinopeptide A was associated with high C-reactive protein and apolipoprotein(a) and lower ankle-brachial index. There were no significant associations of the thrombin markers with race, fibrinogen, alcohol consumption, diabetes, or most measures of subclinical CVD. Study findings support a hypothesis that there are physiological interrelationships between cardiac risk factors, hemostasis, inflammation, and progression of atherosclerosis.
Effects of CSF-1 on Cholesterol Accumulation and Efflux by Macrophages
Authors: Jessup Wendy , Squires Brett , Kritharides Leonard , Hume David A. , Dean Roger T.
Published: June 2011

To assess whether human monocyte-specific colony-stimulating factor (CSF-1) might influence atherogenesis, CSF-1–induced macrophage responses that might contribute to enhanced clearance of low-density lipoprotein (LDL) or modified LDL were investigated. Careful account was made of cell preservation and increases in cell volume and protein (representing increased cell surface area, and thus endocytically active membrane) during culture with CSF-1. This permitted distinction between selective and nonspecific effects of CSF-1, the latter paralleling increases in cellular mass and volume. CSF-1 enhanced mouse peritoneal macrophage survival in vitro during exposure to lipoprotein-deficient serum with or without native LDL or acetylated LDL (Ac-LDL), as judged by maintenance of cellular DNA and cell numbers. In the presence of copper-oxidized LDL (Ox-LDL), such effects were very slight. In all conditions, CSF-1 increased cellular protein content. CSF-1 increased the uptake of both Ac-LDL and Ox-LDL calculated per culture, but this was entirely explicable by the increased cell protein, indicating that there was no selective enhancement of scavenger receptor or other routes for uptake of the modified LDLs. Similarly, CSF-1 also increased the accumulation of cholesterol and its esters nonspecifically. CSF-1 did have a marked and specific effect on the composition of cholesterol esters, decreasing the proportion of polyunsaturated esters relative to monounsaturated and saturated esters. Finally, cholesterol efflux induced by apolipoprotein A1 from Ac-LDL–loaded macrophages was not influenced by CSF-1. Thus, the enhanced macrophage catabolism of modified LDLs by CSF-1 is part of a nonspecific action on the cells but could contribute to a reduction in circulating cholesterol, observed in some situations of CSF-1 presentation in humans.
Fibrinogen and Its Relations to Subclinical Extracoronary and Coronary Atherosclerosis in Hypercholesterolemic Men
Authors: Levenson Jaime , Giral Philippe , Megnien Jean Louis , Gariepy Jérôme , Plainfosse Marie-Christine , Simon Alain
Published: June 2011

The association between plasma fibrinogen and the presence of carotid, femoral, and aortic plaque (high-resolution B-mode ultrasonography) and coronary calcium deposit (ultrafast computed tomography scanner) was determined in 693 hypercholesterolemic, never-treated men free of previous or current clinical symptoms of cardiovascular disease. The number of subjects with extracoronary disease sites and coronary calcification deposits was significantly higher in the upper than in the lower tertile of fibrinogen. Plasma fibrinogen increased according to the number of diseased sites. The odds ratio of the upper to lower fibrinogen tertile for the presence of arterial lesions was 2.6 (1.7 to 4) for carotid, 2.2 (1.5 to 3.2) for aorta, 2.2 (1.5 to 3.1) for femoral, 1.8 (1.3 to 2.6) for coronary, and 3.6 (2.3 to 6.1) for one of four diseased sites. Adjustment for age, total cholesterol, HDL cholesterol, triglycerides, current smoking, and systolic pressure slightly reduced the association between fibrinogen and atherosclerosis. A synergistic effect between fibrinogen and total cholesterol/HDL cholesterol (TC/HDL) ratio seemed to be operating on atherosclerosis, because nearly all of the individuals (98%) had a diseased site when fibrinogen and TC/HDL tertiles were the highest. This result suggests that fibrinogen is involved in the subclinical phase of extracoronary and coronary atherosclerosis and may potentiate the atherogenic effect of hyperlipidemia.
Factor VII, Cholesterol, and Triglycerides
Authors: Green David , Chamberlain Mary Ann , Ruth Karen J. , Folsom Aaron R. , Liu Kiang
Published: June 2011

Cross-sectional studies have shown that factor VII coagulant activity (VII
c
) is positively associated with plasma total cholesterol (TC), LDL cholesterol, and triglycerides (TG) as well as body mass index (BMI) and diastolic blood pressure. To determine whether changes in VII
c
parallel changes in coronary risk factors over a period of 2 years, we examined data from 1514 participants in the Coronary Artery Risk Development in Young Adults Study (CARDIA), an ongoing investigation of lifestyles and evolution of cardiovascular risk factors. Subjects were 23 to 35 years old at the year 5 examination. Cross-sectional analyses at these examinations showed that VII
c
was positively correlated (
P
<.001) with TC and TG in all race/sex groups except for TC in black women at the year 5 examination. Changes in VII
c
over the 2-year period were correlated positively with changes in TC in all except black men and TG in all groups; the association of VII
c
change with change in TC and TG was reduced only slightly with adjustment for age and BMI at year 5 and 2-year change in BMI. To determine whether the higher levels of VII
c
in subjects with higher lipid values were due to activation of the factor or to an increase in the concentration of the factor VII clotting protein, we measured factor VII antigen (VII
ag
) in a randomly selected subsample of 223 subjects at the year 7 examination. In all sex/race groups, VII
ag
correlated with VII
c
(
r
=.69 to 0.81). After adjustment for sex and race, the partial correlation coefficient between TG and VII
c
was .28 (
P
=.0001); between TG and VII
ag
, .35 (
P
=.0001); between TC and VII
c
, .39 (
P
=.0001); and between TC and VII
ag
, 0.43 (
P
=.0001). No associations were observed between lipid levels and the ratio of VII
c
to VII
ag
. We conclude that the raised VII
c
with higher lipid levels occurs in blacks as well as whites, in men and women, persists over time, and represents a true increase in the plasma concentration of this clotting factor.
A Familial Combined Hyperlipidemic Kindred With Impaired Apolipoprotein B Catabolism
Authors: Aguilar-Salinas Carlos A. , Barrett P. Hugh R. , Pulai Judit , Zhu Xin Liang , Schonfeld Gustav
Published: June 2011

Familial combined hyperlipidemia (FCHL) is a heterogeneous disorder characterized by multiple lipoprotein phenotypes, a high risk for coronary heart disease, and predominance among the LDL fraction of smaller and denser particles. We report on an FCHL kindred (the M-kindred) in which decreased VLDL- and LDL-apoB elimination rates rather than enhanced production rates were the main kinetic abnormalities. Lipoprotein levels and metabolic parameters of all apoB-containing lipoproteins (including light and dense LDLs) were determined during placebo and pravastatin treatment periods. ApoB metabolism was studied by endogenous labeling with stable isotopes and a multicompartmental model. Five members of the M-kindred participated. The study was doubly blinded, randomized, and placebo controlled. Treatment periods of 6 weeks were separated by 2-week washout periods. All subjects had high apoB levels, 2 had a mixed lipemia, 1 had hypercholesterolemia, and 2 had hypertriglyceridemia. Familial dysbetalipoproteinemia, hypercholesterolemia, and defective apoB-100 were excluded by genetic testing. Kinetic parameters were remarkably similar in the five study subjects during the placebo period, despite their diverse plasma lipid profiles. Compared with nine normolipidemic control subjects, low VLDL-apoB fractional catabolic rates (FCRs) (3.6±.1 versus 9.3±2.9 pools per day) and low LDL-apoB FCRs (0.19±0.05 versus 0.41±0.13 pool per day) were observed in every case. The majority of the LDL particles were identified in the denser fraction (d=1.036 to 1.063 g/mL). A clear precursor-product relationship was observed from VLDL to IDL to light LDL to dense LDL, ie, there was no “metabolic channeling.” Light LDL had significantly higher FCR than dense LDL (0.82±0.21 versus 0.22±0.08 pool per day). VLDL-apoB production rates were normal (19.7±6.0 versus 21.6±6.1 mg/kg per day for control subjects). In contrast, in two subjects drawn from two other FCHL kindreds (the C- and K-kindreds), VLDL-apoB production rates were increased (35.6 and 32.1 mg/kg per day, respectively). In these two, more “typical” FCHL subjects, FCRs of LDL-apoB were near normal (0.351 and 0.311 pool per day, respectively). Pravastatin (20 mg/d) resulted in significantly lower plasma cholesterol (265±30 to 218±16 mg/dL,P<.01), LDL cholesterol (186±31 to 145±15 mg/dL,P<.03), and apoB levels (168±14 to 125±16 mg/dL,P<.01) in the five FCHL subjects of the M-kindred. No changes were observed in plasma HDL cholesterol, apoA-I, or lipoprotein(a). Pravastatin significantly increased the LDL-apoB FCR (from 0.19±0.05 to 0.34±0.04 pool per day). The FCRs of both LDL subclasses increased with treatment. No pravastatin-induced changes were seen in apoB production rates.
Apolipoprotein A-I
Fin
Authors: Miettinen Helena E. , Gylling Helena , Miettinen Tatu A. , Viikari Jorma , Paulin Lars , Kontula Kimmo
Published: June 2011

We have identified a large kindred with severe serum HDL cholesterol deficiency. The proband, a 65-year-old woman, had greatly diminished concentrations of serum HDL cholesterol (0.19 mmol/L) and apolipoprotein (apo) A-I (21.9 mg/dL). HDL cholesterol and apo A-I levels were similarly reduced in all affected family members, while apo A-II levels were about half of those in the nonaffected family members. Pedigree analysis suggested a dominant inheritance pattern of the phenotype. Sequence analysis of the exons and exon-intron boundaries of the apo A-I gene revealed heterozygosity for a single T-to-G point mutation substituting arginine for leucine at residue 159 of the mature apo A-I protein (apo A-I
Fin
). The T-to-G substitution destroys an
Fsp
I cleavage site, permitting direct polymerase chain reaction/restriction enzyme analysis of the mutation. All the affected family members were shown to be heterozygous for the apo A-I
Fin
mutation. Isoelectric focusing revealed the presence of the mutant apo A-I
Fin
protein in both serum and HDL of the affected subjects. Functional consequences of the mutation were examined by expressing the mutated and wild-type apo A-I cDNAs in COS-7 cells. The mutant apo A-I mRNA had a size similar to that of the normal mRNA, and both mutant and wild-type apo A-I proteins were secreted into the cell media. In vivo kinetic studies of apo A-I revealed increased catabolism in affected subjects. In conclusion, we describe a novel point mutation of the apo A-I gene, apo A-I
Fin
, causing a dominantly negative phenotype as regards serum HDL levels, possibly due to increased catabolism of apo A-I.
Nitric Oxide Synthases and Cardiac Muscle
Authors: Balligand Jean-Luc , Cannon Paul J.
Published: June 2011

AbstractThe different cell types comprising cardiac muscle express one or more of the three isoforms (neuronal NOS, or nNOS; inducible NOS, or iNOS; and endothelial NOS, or eNOS) of nitric oxide synthase (NOS). nNOS is expressed in orthosympathetic nerve terminals and regulates the release of catecholamines in the heart. eNOS constitutively expressed in endothelial cells inhibits contractile tone and the proliferation of underlying vascular smooth muscle cells, inhibits platelet aggregation and monocyte adhesion, promotes diastolic relaxation, and decreases O2consumption in cardiac muscle through paracrinally produced NO. eNOS is also constitutively expressed in cardiac myocytes from rodent and human species, where it autocrinally opposes the inotropic action of catecholamines after muscarinic cholinergic and β-adrenergic receptor stimulation. iNOS gene transcription and protein expression are induced in all cell types after exposure to a variety of inflammatory cytokines. Aside from participating in the immune defense against intracellular microorganisms and viruses, the large amounts of NO produced autocrinally or paracrinally mediate the vasoplegia and myocardial depression characteristic of systemic immune stimulation and promote cell death through apoptosis. In cardiac myocytes, NO may regulate L-type calcium current and contraction through activation of cGMP-dependent protein kinase and cGMP-modulated phosphodiesterases. Other mechanisms independent of cGMP elevations may operate through interaction of NO with heme proteins, non−heme iron, or free thiol residues on target signaling proteins, enzymes, or ion channels. Given the multiplicity of NOS isoforms expressed in cardiac muscle and of the potential molecular targets for the NO produced, tight molecular regulation of NOS expression and activity at the transcriptional and posttranscriptional level appear to be needed to coordinate the many roles of NO in heart function in health and disease.
Nitric Oxide Mediates LDL Uptake in the Artery Wall in Response to High Concentrations of 17β-Estradiol
Authors: Roberts Kim A. , Woo Mable M. , Rutledge John C.
Published: June 2011

Abstract
Female sex hormones are known to affect lipoprotein flux in the artery wall and atherosclerosis. However, the mechanisms of these artery wall effects are unclear. To examine the effect of 17β-estradiol (estradiol) on LDL uptake in the artery wall, we developed an isolated perfused rat carotid artery model from ovariectomized rats. LDL flux in the artery wall was measured by quantitative fluorescence microscopy before and after treatment with estradiol (0.001 to 10 000 nmol/L). Dose-response experiments showed no significant difference in the rate of LDL uptake when arteries were perfused with estradiol at physiological concentrations (0.001 to 1 nmol/L) compared with control perfusions. However, higher concentrations of estradiol (10 to 10 000 nmol/L) significantly increased the rate of LDL uptake in isolated arteries. Artery lumen volume significantly increased with perfusion of estradiol (1 to 100 nmol/L) but decreased after perfusions of higher concentrations of estradiol (1000 to 10 000 nmol/L). Additional studies were performed to examine mechanisms of estradiol-mediated increases in LDL uptake. The effect of estradiol (10 nmol/L) on the rate of LDL uptake was blocked by nitric oxide synthase inhibitors. However, the estrogen receptor antagonist tamoxifen did not block the effects of estradiol on the rate of LDL uptake. Our study indicates that modulation of LDL uptake in the artery wall by estradiol is concentration dependent. High concentrations of estradiol increase LDL uptake by production of endothelium-derived nitric oxide. These observations suggest that increased nitric oxide production compromises endothelial layer barrier function to increase LDL uptake in the artery wall.
Gender-Specific Differences in the Effects of Testosterone and Estrogen on the Development of Atherosclerosis in Rabbits
Authors: Bruck Birgit , Brehme Ute , Gugel Natalia , Hanke Sybille , Finking Gerald , Lutz Caroline , Benda Norbert , Schmahl Friedrich W. , Haasis Rainer , Hanke Hartmut
Published: June 2011

Abstract
The aim of the present study was to investigate whether there are gender-specific differences in the effects of testosterone and estrogen on the process of atherogenesis. Thirty-two castrated male and 32 ovariectomized female rabbits were separated into 4 study groups of 8 males and 8 females each and received postoperatively a 0.5% cholesterol diet for 12 weeks. During this period either no hormones, estradiol (1 mg/kg body wt/week), testosterone (25 mg/kg body wt/week IMM), or estrogen combined with testosterone in above dosages were administered. Computerized morphometric analysis of the intimal thickening in the proximal aortic arch showed a significant inhibitory effect of estrogen in female and of testosterone in male animals (
P
<.05). In the group with combined treatment, the plaque size in both sexes was smaller than in the animals of the control group (
P
<.05). These differences were independent of changes in plasma lipid parameters. The incorporation of 5′-bromo-2′-deoxyuridine, associated with cell proliferation, into cells of the neointima was not significantly affected by the different hormone application regimens in males. In females, the incorporation rate was significantly lowered in the estrogen treated group compared with the control group (
P
<.05). Due to the observed differences in the sex specific atheroprotective effects of testosterone and estrogen, these data suggest that complex hormone interactions, which are independent of changes in plasma lipids, may play an important role in the process of atherogenesis.
Fas Is Expressed in Human Atherosclerotic Intima and Promotes Apoptosis of Cytokine-Primed Human Vascular Smooth Muscle Cells
Authors: Geng Yong-Jian , Henderson Lynne E. , Levesque Ethan B. , Muszynski Maria , Libby Peter
Published: June 2011

AbstractThe membrane protein Fas/Apo-1/CD95 signals programmed cell death or apoptosis in activated T lymphocytes. Vascular smooth muscle cells (SMCs) bear markers of programmed cell death or apoptosis in advanced atherosclerotic plaques that contain immune cells eg, macrophages and T lymphocytes. This study tested the hypothesis that the Fas death-signaling pathway contributes to apoptosis of SMCs exposed to proinflammatory cytokines produced by these immune cells during atherogenesis. All atherosclerotic plaques examined (n=14) contained immunoreactive Fas. The majority of the Fas+SMCs localized in the intima of the plaques, whereas the medial SMCs expressed Fas antigen less prominently. Double staining for DNA fragments (TUNEL) and Fas or cell identification markers colocalized Fas with TUNEL+SMCs in the areas that contained CD3+T cells and CD68+macrophages, suggesting a role for Fas in the induction of SMC apoptosis by activated T cells during atherogenesis. In culture, stimulation with interferon-γ, tumor necrosis factor-α, and interleukin-1β increased expression of Fas in SMCs. Incubation with an activating anti-Fas antibody triggered apoptosis of the cytokine-primed but not the untreated SMCs, as demonstrated by TUNEL and electrophoresis of oligonucleosomal DNA fragments. These data suggest that activation of the Fas death-signaling pathway contributes to the induction of SMC apoptosis during atherogenesis and furnish a mechanism whereby immune cells and their cytokines promote this cell death process related to vascular remodeling and plaque rupture.
Remodeling With Neointima Formation in the Mouse Carotid Artery After Cessation of Blood Flow
Authors: Kumar Anjali , Lindner Volkhard
Published: June 2011

Abstract
The ability of gene targeting in the mouse species presents a powerful tool to determine the role of specific molecules in vascular biology. Using a denuding-injury procedure, we recently reported that intimal lesions can be induced in the carotid artery of outbred mice. The technical challenge associated with achieving complete denudation and the relatively small size of the developing lesions prompted us to design the present model of neointima formation and vascular remodeling in the carotid artery of the inbred FVB mouse strain. Complete ligation of the vessel near the carotid bifurcation induced rapid proliferation of medial smooth muscle cells, leading to extensive neointima formation in the presence of an endothelial lining. Thrombus formation was not observed except in the most distal part of the vessel adjacent to the ligature. At 4 weeks after ligation, luminal area was reduced by ≈80% through a combination of decreased vessel diameter and neointima formation. Ultrastructural analysis provided evidence for cell death in the developing neointima as well as the remodeling media. The present model might be useful in identifying those genes important for neointima formation and vascular remodeling.
Evidence for an α-Granular Pool of the Cytoskeletal Protein α-Actinin in Human Platelets That Redistributes With the Adhesive Glycoprotein Thrombospondin-1 During the Exocytotic Process
Authors: Dubernard Véronique , Arbeille Brigitte B. , Lemesle Monique B. , Legrand Chantal
Published: June 2011

Abstract
In a previous study, we have demonstrated that the platelet adhesive glycoprotein thrombospondin-1 (TSP-1) interacts specifically with the cytoskeletal protein α-actinin in a solid-phase binding assay. Stored in the α-granules of platelets, TSP-1 is secreted during cell activation and binds to the plasma membrane promoting the platelet macroaggregate formation. However, the molecular mechanism by which TSP-1 reaches and binds to the platelet surface is to date unelucidated. α-Actinin is an actin-binding and actinin–cross-linking protein that is present in most cells and may act as a link between the bundles of F-actin and the plasma membrane. In this study, we have investigated a possible interaction of α-actinin with TSP-1 in platelets by examining their respective subcellular location during the platelet activation process. By indirect immunofluorescence, α-actinin was found to display a granular staining in resting platelets similar to that of TSP-1. Performing postembedding immunogold labeling for electron microscopy, we detected the presence of α-actinin throughout the cytoplasm, but the strongest gold staining was found in organelles identified as α-granules on the basis of their ultrastructure and TSP-1 content. With the use of double immunogold labeling on platelets at different stages of activation by thrombin, both α-actinin and TSP-1 were seen redistributing from the α-granules to the platelet surface via the open canalicular system (OCS). At the same time, the cytoplasmic α-actinin concentrated toward the plasma membrane, but no colocalization with the F-actin bundles was evidenced. Finally, preembedding immunogold labeling and immunoprecipitation of
125
I-surface–labeled, thrombin-activated platelets further demonstrated that α-actinin was expressed on the plasma membrane in the absence of any detectable expression of actin and that it could form molecular complexes with TSP-1 on activated platelets. These results suggest that α-actinin found to be present on the platelet surface together with TSP-1 originates in the α-granules by fusion of the α-granules with the plasma membrane during platelet exocytosis.
Short-term Exposure to Thapsigargin Inhibits Neointima Formation in Human Saphenous Vein
Authors: George S. J. , Johnson J. L. , Angelini G. D. , Jeremy J. Y.
Published: June 2011

Abstract
Vascular smooth muscle cell (VSMC) migration and proliferation are involved in the intimal thickening responsible for late vein graft failure. In addition to growth and chemotactic factors, VSMCs require expression of matrix-degrading enzymes, eg, metalloproteinases (MMP), to relieve the antiproliferative and antimigratory constraints of the extracellular matrix. Thapsigargin irreversibly inhibits Ca
2+
- ATPase, eliciting an increase in intracellular Ca
2+
and depletion of the intracellular calcium pools that are thought to be involved in the control of VSMC migration, VSMC proliferation, and MMP activity. We therefore studied the effect of thapsigargin on VSMC migration, VSMC proliferation, and MMP expression in human saphenous vein organ cultures. Vein segments were cultured for 14 days, and VSMC proliferation and migration were determined by autoradiography. Cell death was assessed using in situ end-labeling and lactate dehydrogenase release. Using Western blotting, we examined MMP-2 and MMP-9 and tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 expression. Exposure to thapsigargin at 10 nmol/L for 60 minutes before culture significantly inhibited neointimal thickening (60%,
P
<.05), intimal and medial VSMC proliferation (32%,
P
<.05 and 37%,
P
<.05, respectively), and VSMC migration (36%,
P
<.05). Thapsigargin at 10 nmol/L did not significantly increase cell death or MMP-2, MMP-9, TIMP-1, and TIMP-2 expression. These results suggest that blockade of Ca
2+
-ATPase by thapsigargin inhibits VSMC migration and proliferation involved in neointimal formation without affecting MMP-2 and MMP-9 expression. Because short-term exposure to thapsigargin was sufficient to inhibit neointima formation, this drug may prove useful in the treatment of intimal thickening after arterial bypass graft surgery.
Platelet-Derived Growth Factor β-Receptors Can Both Promote and Inhibit Chemotaxis in Human Vascular Smooth Muscle Cells
Authors: Clunn Gerard F. , Refson Jonathan S. , Lymn Joanne S. , Hughes Alun D.
Published: June 2011

AbstractThe effect of the three platelet-derived growth factor (PDGF) isoforms AA, AB, and BB on migration was investigated in cultured human saphenous vein smooth muscle cells. The modified Boyden chamber technique yielded efficacies BB>>AB, AA=0. However, the BB concentration-response relationship displayed a pronounced peak, occurring between 1 and 10 ng/mL, with no response above this range. Checkerboard analysis showed that the promotion of migration at low concentrations was chemotactic in nature but that the downturn was independent of gradient. Furthermore, at high concentrations BB was able to prevent chemotaxis induced by fetal calf serum and epidermal growth factor (EGF). Experiments using low concentrations of BB in combination with high concentrations of AA to saturate PDGF α-receptors in the presence and absence of a neutralizing antibody to α-receptors revealed that α-receptor activation induced partial inhibition of chemotaxis but this did not account for the inhibition of migration by high concentrations of BB. Despite possessing no significant chemotactic action itself, high concentrations of the AB isoform completely inhibited BB induced chemotaxis. Taken together these results suggest that the chemotactic signal induced by PDGF is dominated by PDGF β-receptors and switches from positive at low concentrations to negative at higher concentrations. Stimulation of DNA synthesis by the three isoforms (as measured by [3H] thymidine incorporation) yielded saturable responses for the AB and BB isoforms, with similar efficacy and weak or no response for the AA isoform. Concentration-dependent patterns of tyrosine phosphorylation of certain proteins mirrored the form of the chemotactic response and suggest one possible underlying regulatory mechanism to account for the disparity between PDGF-induced chemotaxis and DNA synthesis.
Upregulation of Low Density Lipoprotein Receptor by Gemfibrozil, a Hypolipidemic Agent, in Human Hepatoma Cells Through Stabilization of mRNA Transcripts
Authors: Goto Daisuke , Okimoto Tadayoshi , Ono Mayumi , Shimotsu Hidenori , Abe Kazuhiro , Tsujita Yoshio , Kuwano Michihiko
Published: June 2011

Abstract
Gemfibrozil reduces the plasmal levels of cholesterol and triglyceride in patients with hyperlipidemia by a mechanism that is not well understood. The present study evaluated the effect of gemfibrozil on the LDL receptor in human hepatoma cells compared with that of pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Exposure to gemfibrozil, 40 μmol/L, for 3 days increased the binding of
125
I-LDL to the surface of three lines of human hepatoma cell, HepG2, HuH7, and HLE by 1.5- to 2.0-fold. Similar findings were observed with pravastatin. Scatchard analysis with
125
I-LDL indicated an increased number of LDL receptors on the cell surface of HepG2 cells when treated with gemfibrozil and pravastatin. However, the gemfibrozil-treated cells exhibited no increase in the binding of
125
I-epidermal growth factor (EGF). Gemfibrozil increased the levels of LDL receptor mRNA and protein in HepG2 cells. The increase in LDL receptor activity induced by pravastatin was abolished by concomitant administration of mevalonic acid, 770 μmol/L. This effect was not seen with gemfibrozil, suggesting the mechanism differs for the two lipid-lowering drugs. To determine whether this increase in mRNA was due to transcriptional activation, we prepared HepG2 cells transfected with an LDL receptor promoter-reporter construct that contained a sterol regulatory element. The expression of LDL receptor regulated by the sterol regulatory element was increased by pravastatin, but not by gemfibrozil. We evaluated the stability of the mRNA in the presence of actinomycin D to explain the increase in the LDL receptor mRNA. Gemfibrozil prolonged the half-life of the mRNA for LDL receptor but not that for the EGF receptor. Stabilization of the LDL receptor mRNA is suggested to be the novel mode of action of gemfibrozil.
Lipoprotein(a) Interactions With Lipid and Nonlipid Risk Factors in Early Familial Coronary Artery Disease
Authors: Hopkins Paul N. , Wu Lily L. , Hunt Steven C. , James Brent C. , Vincent G. Michael , Williams Roger R.
Published: June 2011

AbstractAn interaction between high plasma lipoprotein(a) [Lp(a)], unfavorable plasma lipids, and other risk factors may lead to very high risk for premature CAD. Plasma Lp(a), lipids, and other coronary risk factors were examined in 170 cases with early familial CAD and 165 control subjects to test this hypothesis. In univariate analysis, relative odds for CAD were 2.95 (P<.001) for plasma Lp(a) above 40 mg/dL. Nearly all the risk associated with elevated Lp(a) was found to be restricted to persons with historically elevated plasma total cholesterol (6.72 mmol/L [260 mg/dL] or higher) or with a total/HDL cholesterol ratio >5.8. Nonlipid risk factors were also found to at least multiply the risk associated with Lp(a). When Lp(a) was over 40 mg/dL and plasma total/HDL cholesterol >5.8, relative odds for CAD were 25 (P=.0001) in multiple logistic regression. If two or more nonlipid risk factors were also present (including hypertension, diabetes, cigarette smoking, high total homocysteine, or low serum bilirubin), relative odds were 122 (P<1×10−12). The ability of nonlipid risk factors to increase risk associated with Lp(a) was dependent on at least a mildly elevated total/HDL cholesterol ratio. In conclusion, high Lp(a) was found to greatly increase risk only if the total/HDL cholesterol ratio was at least mildly elevated, an effect exaggerated by other risk factors. Aggressive lipid lowering in those with elevated Lp(a) therefore appears indicated.
Genetic Factors Precipitating Type III Hyperlipoproteinemia in Hypolipidemic Transgenic Mice Expressing Human Apolipoprotein E2
Authors: Huang Yadong , Rall Stanley C. , Mahley Robert W.
Published: June 2011

Abstract
Several factors are hypothesized to precipitate or exacerbate type III hyperlipoproteinemia (HLP) in humans. Among such factors are those that directly overload remnant lipoprotein production or disrupt removal pathways, including an increased ratio of apolipoprotein (apo) E2 to normal apoE, overproduction of apoB-containing lipoproteins, and decreased LDL receptor activity. Hypolipidemic apoE2-transgenic mice bred onto an apoE-null background had dramatically higher plasma total cholesterol (192±26 mg/dL for males, 203±40 mg/dL for females) and triglyceride (295±51 mg/dL for males, 277±58 mg/dL for females) levels than apoE2 mice with endogenous mouse apoE. Thus, eliminating normal apoE in the presence of apoE2 (thereby increasing the relative abundance of the defective ligand) can convert a hypolipidemic to a hyperlipidemic phenotype. Hypolipidemic apoE2 transgenic mice overexpressing human apoB had moderate remnant accumulation compared with apoE2-only or apoB-only transgenic mice, indicating that overproduction of apoB-containing lipoproteins in the presence of apoE2 can augment remnant production. Hypolipidemic apoE2 transgenic mice bred onto an LDL receptor–null background had markedly higher plasma total cholesterol (288±51 mg/dL for males, 298±73 mg/dL for females) and triglyceride (356±72 mg/dL for males, 317±88 mg/dL for females) levels than apoE2-only mice, and remnant accumulation increased even in apoE2 mice with a heterozygous LDL receptor–knockout background (compared with apoE2-only mice), suggesting that reducing or eliminating a major receptor-mediated remnant-removal pathway in the presence of apoE2 can also precipitate a hyperlipidemic phenotype. In all cases where either lipoprotein remnant production or removal pathways were severely stressed, increased remnant accumulation was apparent. As judged by the chemical characteristics of the remnant lipoproteins, the lipoprotein phenotype was quite similar to that of human type III HLP, especially in the apoE2-expressing mice with no endogenous apoE or LDL receptors, and thus these mice represent improved models of the disorder.
In Vivo Glucosylated LpA-I Subfraction
Authors: Igau Bruno , Castro Graciela , Clavey Véronique , Slomianny Christian , Bresson Régis , Drouin Pierre , Fruchart Jean-Charles , Fiévet Catherine
Published: June 2011

Abstract
This study compared the structural and functional properties of glucosylated and non-glucosylated LpA-I particle subfractions (GLpA-I and NGLpA-I, respectively) isolated from patients with poorly controlled type 1 (insulin-dependent) diabetes. Compared with NGLpA-I, GLpA-I showed an enrichment in triglycerides (
P
<.05) and a depletion in phospholipid (
P
<.05) content. Moreover, the triglycerides-to-cholesteryl esters ratio was increased (
P
<.05), suggesting an increased cholesteryl ester transfer protein activity and a possible transport defect that accelerates atherogenesis. The surface-to-core constituents ratio, an indirect estimate of particles size, is lower in GLpA-I (
P
<.01) than in NGLpA-I, correlating well with a larger median size (
P
<.05) as seen by electron microscopy. The apolipoprotein (apo) A-I conformation was evaluated through determination of the immunological accessibility of three different domains defining specific epitopes for anti-apo A-I monoclonal antibodies. We observed a marked decreased accessibility for two of these regions, which interestingly have already been implicated in the interaction with cells. Cell culture data suggest that nonenzymatic glycosylation occurring on apo A-I can modify lipoprotein function, since it results in a decreased binding of GLpA-I to HeLa cells and impaired cholesterol efflux from Fu5AH rat hepatoma cells.
Paracetamol Catalyzes Myeloperoxidase-Initiated Lipid Oxidation in LDL
Authors: Kapiotis S. , Sengoelge G. , Hermann M. , Held I. , Seelos C. , Gmeiner B. M. K.
Published: June 2011

Abstract
The oxidative modification of LDL may play a significant role in atherogenesis. Myeloperoxidase (MPO) expressed in human atherosclerotic plaques has been suggested to be operative in vivo, making LDL atherogenic. Tyrosyl radicals generated by MPO have been shown to act as physiological pro-oxidants of lipid peroxidation in LDL. Assuming that a variety of phenolic compounds are able to form phenoxyl radicals when exposed to peroxidases, we tested the ability of paracetamol, a known analgesic drug with a tyrosine-like monophenolic structure, to act as a pro-oxidant of lipid peroxidation in LDL. Spectroscopic analyses indicated that paracetamol, similar to tyrosine, could undergo peroxidase-induced phenoxyl radical formation, which was inhibited by the radical scavenger ascorbic acid as well as by heme poisons and catalase. Measurement of conjugated dienes and lipid hydroperoxides in LDL preparations exposed to MPO/H
2
O
2
in the absence or presence of paracetamol revealed that the drug could act as a catalyst of lipid oxidation in LDL. Similar results were found when LDL oxidation was performed with activated human neutrophils, which use MPO to promote lipid peroxidation. In conclusion, the results suggest that paracetamol could act, via a phenoxyl radical, as a catalyst of LDL oxidative modification by MPO.
Effects of Dietary Fat Quality and Quantity on Postprandial Activation of Blood Coagulation Factor VII
Authors: Larsen Lone Frost , Bladbjerg Else-Marie , Jespersen Jørgen , Marckmann Peter
Published: June 2011

Abstract
Acute elevation of the coagulant activity of blood coagulation factor VII (FVIIc) is observed after consumption of high-fat meals. This elevation is caused by an increase in the concentration of activated FVII (FVIIa). In a randomized crossover study, we investigated whether saturated, monounsaturated, or polyunsaturated fats differed regarding postprandial activation of FVII. Eighteen healthy young men participated in the study. On 6 separate days each participant consumed two meals (times, 0 and 13/4 hours) enriched with 70 g (15 and 55 g) of either rapeseed oil, olive oil, sunflower oil, palm oil, or butter (42% of energy from fat) or isoenergetic low-fat meals (6% of energy from fat). Fasting and series of nonfasting blood samples (the last at time 81/2 hours) were collected. Plasma triglycerides, FVIIc, FVIIa, and free fatty acids were analyzed. There were marked effects of the fat quantity on postprandial responses of plasma triglycerides, FVII, and free fatty acids. The high-fat meals caused, in contrast to the low-fat meals, considerable increases in plasma triglycerides. Plasma levels of FVIIc and FVIIa peaks were 7% and 60% higher after consumption of high-fat meals than after consumption of low-fat meals. The five different fat qualities caused similar postprandial increases in plasma triglycerides, FVIIc, and FVIIa. These findings indicate that high-fat meals may be prothrombotic, irrespective of their fatty acid composition. The postprandial FVII activation was not associated with the plasma triglyceride or free fatty acid responses.
Thrombomodulin Expression in Bovine Brain Capillaries
Authors: Wang Liang , Tran Nam D. , Kittaka Mamoru , Fisher Mark J. , Schreiber Steven S. , Zlokovic Berislav V.
Published: June 2011

AbstractThrombomodulin (TM), a key cofactor of the TM-protein C pathway, is of major biologic significance for the antithrombotic properties of endothelial cells. Yet, there is uncertainty whether TM is expressed in brain and what mechanisms govern brain endothelial anticoagulant activity. In this study, bovine brain capillaries were used as an in vitro model of the blood-brain barrier to determine factors involved in the regulation of TM expression in cerebral vasculature. Quantitative competitive-polymerase chain reaction assay revealed significant regional differences in the amount of brain capillary TM mRNA, ie, cortical > cerebellar > pontine, consistent with the reverse transcription-polymerase chain reaction findings in which the abundance of TM mRNA was analyzed relative to β-actin mRNA. Regional differences in TM mRNA brain capillary level correlated well with differences in protein C activation. The TM mRNA and activity were not detectable in brain parenchyma. Pathogenic mediators of ischemic stroke, interleukin 1β (10 U/mL), and tumor necrosis factor α (10 U/mL), produced a time-dependent decrease in brain capillary TM mRNA (t1/2of 2.1 and 3.9 hours, respectively) and reduced endothelial TM activity. Incubation of brain capillaries with retinoic acid (10 μmol/L) and dibutyryl cAMP (3 mmol/L) resulted in a 4-fold increase in TM mRNA at 4 and 8 hours, respectively, followed by an increase in protein C activation. We conclude that TM at the blood-brain barrier is likely to be an important physiologic anticoagulant in brain microcirculation. Its downregulation by cytokines may contribute to ischemic brain damage and potentially could be counteracted by retinoic acid and cAMP.