bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of Mitogen-Activated Protein Kinases (ERK/JNK) and AP-1 Transcription Factor in Rat Carotid Arteries After Balloon Injury
Authors: Hu Yanhua , Cheng Linda , Hochleitner Boris-Wolfgang , Xu Qingbo
Published: June 2011

Abstract
Smooth muscle cell proliferation is a key event in neointimal formation after balloon angioplasty. The molecular signals that mediate this process have yet to be identified. Mitogen-activated protein (MAP) kinases are thought to play a pivotal role in transmitting transmembrane signals required for cell proliferation in vitro. The present studies were designed to investigate whether the signal transduction pathways of MAP kinases were involved in the development of restenosis in the injured arteries. Rat carotid arteries were isolated at various time points after balloon injury, and activities of MAP kinases, including extracellular signal-regulated kinases (ERK), and stress activated protein kinases (SAPK)/c-Jun N-terminal protein kinases (JNK), were determined in protein extracts of the vasculature using protein kinase assay and Western blot analysis. After balloon angioplasty, ERK2 and JNK1 activities in the vessel wall increased rapidly, reached a high level in 5 minutes and maintained for 1 hour. A sustained increase in ERK2 kinase activity was observed over the next 7 days in the arterial wall and 14 days in neointima after injury. In contrast, opposite and uninjured arteries did not show significant changes in these kinase activities. Concomitantly, Western blot analysis confirmed that the ERK2 kinase in the injured vessels was indeed activated or phosphorylated, showing a slowly migrating species of a 42-kDa protein containing phosphorylated tyrosine. Kinase activation is followed by an increase in
c-fos
and
c-jun
gene expression and enhanced activator protein 1 (AP-1) DNA-binding activity. Thus, balloon injury rapidly activates the MAP kinases in rat carotid arteries. These kinase activations may be crucial in mediating smooth muscle cell proliferation in response to vascular angioplasty.
Evidence Against an Effect of Endothelin-1 on Blood Coagulation, Fibrinolysis, and Endothelial Cell Integrity in Healthy Men
Authors: Kapiotis Stylianos , Jilma Bernd , Szalay Tivadar , Dirnberger Eva , Wagner Oswald , Eichler Hans-Georg , Speiser Wolfgang
Published: June 2011

Abstract
On the basis of an array of preclinical experimental results, it has been widely assumed that endothelin-1 (ET-1) may affect blood coagulation, fibrinolysis, and endothelial cell function, thereby playing a pathophysiological role in various cardiovascular diseases in humans. However, confirmation of this assumption is still lacking. ET-1 or placebo was administered intravenously to 12 healthy volunteers in a prospective, randomized, double-blind, crossover trial. Pathophysiologically relevant concentrations of ET-1 (an approximate threefold increase of normal blood levels) causing hemodynamic effects were reached by continuous intravenous infusion for 6 hours. Components of the coagulation (thrombin-antithrombin complexes, prothrombin fragment F
1+2
, activated factor VII, and factor VII antigen) and fibrinolytic (fibrin split product D-dimer, plasmin–plasmin inhibitor complex, tissue-type plasminogen activator, urokinase-type plasminogen activator, and plasminogen activator inhibitor-1) systems and markers of endothelial cell perturbation/dysfunction (von Willebrand factor and thrombomodulin) were measured before the start of infusion and after 2, 6, 12, and 24 hours. Comparing changes in the plasma concentrations of these parameters during and after infusion of ET-1 and placebo, we found no specific effects of ET-1. In contrast to previous reports from preclinical experiments, ET-1 does not appear to affect coagulation or fibrinolysis, nor does this peptide induce relevant endothelial cell perturbations in humans.
Evidence for the Migration of Rat Aortic Endothelial Cells Toward the Heart
Authors: Kiosses W. B. , McKee N. H. , Kalnins V.I.
Published: June 2011

Abstract
Most vascular endothelial cells at the edge of experimentally induced wounds have their centrosomes oriented toward the wound in the direction of cell migration. The finding that the centrosomes in endothelial cells of nonwounded aorta and vena cava are also oriented toward the heart suggested the hypothesis that endothelial cells are normally migrating in this direction. To test this hypothesis, endothelial cells in a segment of the rat abdominal aorta were labeled with a relatively nontoxic dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), and the position of the labeled cells was determined 3 and 6 weeks later. The results obtained showed that in 6 of the 9 rat aortas examined at 3 weeks and 15 of the 20 rat aortas examined at 6 weeks, DiI-labeled endothelial cells had migrated various distances up to 5000 μm toward the heart. In contrast, no migration of endothelial cells was detected at the opposite end of the labeled segment, in the direction away from the heart. These results demonstrate that vascular endothelial cells in the abdominal aorta of the rat are not stationary but are migrating toward the heart. The significance of the migration of endothelial cells toward the heart is presently unknown; however, it would be interesting to explore whether or not the impairment of this migration may contribute to disease processes in which the ability to maintain an intact and normally functioning endothelial cell lining is compromised as in atherosclerosis.
ApoE Genotype Does Not Predict Lipid Response to Changes in Dietary Saturated Fatty Acids in a Heterogeneous Normolipidemic Population
Authors: Lefevre Michael , Ginsberg Henry N. , Kris-Etherton Penny M. , Elmer Patricia J. , Stewart Paul W. , Ershow Abby , Pearson Thomas A. , Roheim Paul S. , Ramakrishnan Rajasekhar , Derr Janice , Gordon David J. , Reed Roberta , for the DELTA Research Group
Published: June 2011

Abstract
Recent studies have suggested that variations in apoE genotypes may influence the magnitude of plasma lipid changes in response to dietary interventions. We examined the ability of apoE genotype to predict plasma lipid response to reductions in percent of calories from total fat (TF) and saturated fat (SF) in a normolipidemic study population (n=103) heterogeneous with respect to age, gender, race, and menopausal status. Three diets, an average American diet (34.3% TF, 15.0% SF), an AHA Step 1 diet (28.6% TF, 9.0% SF), and a low saturated fat (Low-Sat) diet (25.3% TF, 6.1% SF) were each fed for a period of 8 weeks in a three-way crossover design. Cholesterol was kept constant at 275 mg/d; monounsaturated and polyunsaturated fat were kept constant at approximately 13% and 6.5% of calories, respectively. Fasting lipid levels were measured during each of the final 4 weeks of each diet period. Participants were grouped by apoE genotype: E2 (E2/2, E2/3, E2/4); E3 (E3/3); E4 (E3/4, E4/4). Relative to the average American diet, both the Step 1 and Low-Sat diets significantly reduced total cholesterol, LDL cholesterol, and HDL cholesterol in all three apoE genotype groups. No evidence of a significant diet by genotype interaction, however, could be identified for any of the measured lipid and lipoprotein end points. Additional analysis of the data within individual population subgroups (men and women, blacks and whites) likewise provided no evidence of a significant diet by genotype interaction. Thus, in a heterogeneous, normolipidemic study population, apoE genotype does not predict the magnitude of lipid response to reductions in dietary saturated fat.
Hyperhomocyst(e)inemia and a Common Methylenetetrahydrofolate Reductase Mutation (Ala
223
Val MTHFR) in Patients With Inherited Thrombophilic Coagulation Defects
Authors: Legnani Cristina , Palareti Gualtiero , Grauso Francesca , Sassi Simonetta , Grossi Gabriele , Piazzi Sandro , Bernardi Francesco , Marchetti Giovanna , Ferraresi Paolo , Coccheri Sergio
Published: June 2011

Abstract
To assess whether certain abnormalities of the sulfated amino acid metabolism are associated with the occurrence of thromboembolic events in patients with inherited thrombophilic conditions, the levels of homocyst(e)ine, before or after methionine load, and the presence of the Ala
223
Val substitution in the 5,10-methylenetetrahydrofolate reductase (MTHFR) were evaluated in 119 subjects with a congenital single thrombophilic condition (type I deficiency of antithrombin n=10, protein C n=24, protein S n=16; activated protein C resistance due to factor V Leiden mutation n=69). Sixty-three subjects had experienced at least one documented thrombotic event, while the remaining 56 subjects were still free from any thrombotic symptom. Our results show that (1) high homocyst(e)ine levels, either in fasting condition or after methionine load, were not more frequent in subjects with inherited thrombophilic alterations (14.4%) than in normal control subjects (10% by definition) and (2) the frequency of hyperhomocyst(e)inemia was similar in thrombophilic subjects, who already have (14.3%) or have not (14.6%) experienced thrombotic events. As regards the MTHFR mutation, the homozygous condition was present in 23.2% of the thrombophilic patients versus 17.5% in the control subjects, a nonsignificant difference. The mutation was slightly more frequent in those thrombophilic subjects who had suffered a thrombotic episode (25.5%) versus those with no thrombosis (20.8%), with odds ratios of 1.61 (confidence interval (CI)=0.58-4.52) and 1.24 (CI=0.42-3.43), respectively. These differences were also nonsignificant. It is concluded that in subjects with inherited thrombophilias, a condition of hyperhomocyst(e)inemia “per se” is not a factor increasing the risk of thrombosis. The risk enhancement conferred by the MTHFR mutation, if any, seems to be slight or limited, and its significance could be ascertained only in a large multicenter trial.
Two Alleles of the Human Paraoxonase Gene Produce Different Amounts of mRNA
Authors: Leviev Ilia , Negro Franco , James Richard W.
Published: June 2011

Abstract
In a recent study we demonstrated that a polymorphism of the human paraoxonase gene affecting position 54 was linked to variations in serum concentrations of the enzyme. L allele carriers (leucine at position 54) have significantly higher concentrations of paraoxonase than M allele carriers (methionine at position 54). In the present study we examined the hypothesis that differences in mRNA production could contribute to variations in serum concentrations. Relative concentrations of L and M type mRNA were analyzed in total RNA extracted from heterozygous liver samples. This was achieved by cDNA synthesis, polymerase chain reaction amplification of the cDNA fragment containing the 54 polymorphism and restriction analysis to identify radiolabeled end fragments of L and M alleles. An allele mixing experiment using total RNA from liver samples of LL and MM homozygotes demonstrated the sensitivity of the approach to changes in the relative concentrations of each type of RNA. In 8 of 10 heterozygous samples, an excess of L allele type mRNA was observed. Overall there was a significantly higher level of L type mRNA (L:M ratio of 2.51±1.41, n=10,
P
<.01). These results support our hypothesis that increased concentrations of serum paraoxonase arise from greater production of L allele mRNA. In two samples, the L:M ratio was close to or below 1.0. This is consistent with the known spectrum of paraoxonase serum concentrations associated with the L and M alleles and suggests that factor(s) that preferentially modulate allele expression are usually, but not uniformly, associated with the L allele.
The Impact of Atherosclerotic Arterial Remodeling on Percentage of Luminal Stenosis Varies Widely Within the Arterial System
Authors: Pasterkamp Gerard , Schoneveld Arjan H. , van Wolferen Willem , Hillen Berend , Clarijs Ruud J. G. , Haudenschild Christian C. , Borst Cornelius
Published: June 2011

Abstract
Luminal stenosis can be based on large atherosclerotic plaques in compensatory enlarged segments or on relatively little plaques in shrunken segments. In the present study, the contribution of plaque formation and remodeling to luminal narrowing was compared among six types of arteries prone to symptomatic atherosclerosis. Cross-sections (n=5195) were obtained at regular intervals from 329 arteries. For each artery, the cross-section that contained the least amount of plaque was considered to be the reference. For each cross-section, the percentage of lumen area decrease was expressed as a percentage of the lumen area at the reference site (luminal stenosis). Similarly, the area encompassed by the internal elastic lamina (IEL area) was expressed as a percentage of the IEL area at the reference site (relative IEL area). All cross-sections were categorized in three groups: relative IEL area >105% (enlargement), 95% to 105% (no remodeling), and <95% (shrinkage). The prevalence of enlargement (50% to 75%) was significantly higher compared with shrinkage (8% to 25%). Shrinkage was observed most frequently in the femoral arteries (25%) and infrequently in the renal arteries (8%). For all types of arteries, the relative IEL area correlated negatively with luminal stenosis (
P
<.001). Regression analysis of relative IEL area on luminal stenosis, however, showed significant differences in the first-order regression coefficients among artery types. On average, plaque increase was more compensated for by enlargement in the coronary, common carotid, and renal arteries compared with the arteries obtained from the lower extremities. Anatomic regional differences were observed in the impact of arterial wall remodeling on percent luminal stenosis in de novo atherosclerotic lesions.
Loss of Arterial Dilation in the Reendothelialized Area of the Flow-Loaded Rat Common Carotid Artery
Authors: Sugiyama Tatsuo , Kawamura Koichi , Nanjo Hiroshi , Sageshima Masato , Masuda Hirotake
Published: June 2011

Abstract
We have investigated regenerated endothelial cells and their possible contribution to arterial dilation in response to increased blood flow in rat common carotid artery (CCA). After endothelial denudation using a balloon catheter in the left CCA, an arteriovenous shunt was constructed between the left CCA and the left external jugular vein at 20 mm distal from the orifice in the denuded group. Animals that were given the arteriovenous shunt without denudation were used to form the nondenuded group. The blood flow rate in the left CCA was increased by sixfold after operation in the denuded group. We observed that endothelial cells were gradually regenerated from the orifice to the distal area and that the reendothelialized area after 4 to 8 weeks was approximately one third of the left CCA (5.31±1.49 mm at 4 weeks, 5.47±1.56 mm at 8 weeks). In the reendothelialized area of the left CCA after 4 to 8 weeks, the lumen diameter was significantly smaller than that of the nondenuded group and showed no significant difference from age-matched nonsurgical animals. The intimal and medial thickening, which would result in arterial stenosis in the reendothelialized area, was not observed in the denuded group, although the denuded control showed significant intimal thickening. From these results, we conclude that regenerated endothelial cells reduce intimal thickening but do not respond to increased blood flow to dilate the artery.
Role of Angiotensin II and Bradykinin on Aortic Collagen Following Converting Enzyme Inhibition in Spontaneously Hypertensive Rats
Authors: Benetos Athanase , Levy Bernard I. , Lacolley Patrick , Taillard François , Duriez Micheline , Safar Michel E.
Published: June 2011

Abstract
We previously showed that chronic angiotensin-converting enzyme (ACE) inhibition prevented the increase in aortic collagen in spontaneously hypertensive rats (SHRs) independently of blood pressure reduction. The aim of the present study was to determine whether the effects of ACE inhibition on aortic fibrosis were due to inhibition of angiotensin II formation, preservation of bradykinin, or a combination of both. Four week-old SHRs were treated for 4 months with the ACE inhibitor quinapril, quinapril with the bradykinin B
2
receptor antagonist Hoe 140, or the angiotensin II AT
1
receptor antagonist CI996. Control SHR and Wistar-Kyoto (WKY) rats received a placebo for the same period of time. At the end of the treatment, as compared to conscious SHR and WKY controls, quinapril completely prevented the development of hypertension, whereas quinapril-Hoe 140 and the AT
1
receptor antagonist produced only a partial reduction of blood pressure. In relation with blood pressure changes, aortic hypertrophy was significantly prevented by quinapril but not by quinapril-Hoe 140 or CI996. In contrast, aortic collagen accumulation was completely prevented by all three treatments. The study provides evidence that in young live SHRs, the prevention of aortic collagen accumulation is independent of blood pressure changes and bradykinin preservation and involves exclusively angiotensin II inhibition through AT
1
receptors.
Deficiency of Cholesteryl Ester Transfer Protein
Authors: Ritsch Andreas , Drexel Heinz , Amann Franz W. , Pfeifhofer Christa , Patsch Josef R.
Published: June 2011

Abstract
A patient is described who exhibited, despite excessively high postprandial triglyceride levels, high levels of HDL cholesterol. Measurement of CETP activity and mass in the patient’s plasma showed values of less than 5% and 2%, respectively, of a normolipidemic plasma pool. The CETP cDNA of the patient exhibited a mutation (T → G), turning codon 57 (TAT) of exon 2 into a stop codon (TAG) and abolishing a,
Xcm
I restriction site. Digestion of directly amplified CETP cDNA from the patient with
Xcm
I indicated the exclusive presence of CETP cDNA containing the mutation. Analysis of the corresponding region of the CETP gene indicated the patient to be heterozygous for the nonsense mutation at codon 57, a finding that can only be explained by the presence of a null allele in addition to the allele with the nonsense mutation. The combination of TG intolerance of uncertain cause, together with CETP deficiency due to a novel mutation, produced the paradoxical constellation—high levels of HDL cholesterol (172 mg/dL) associated with a high postprandial lipemia of 1460 mg triglycerides/dL.8 hours—and provided further insight into the role of CETP as mediator between pools of triglycerides and cholesteryl esters in plasma.
A
192
Arg Variant of the Human Paraoxonase
(HUMPONA)
Gene Polymorphism Is Associated With an Increased Risk for Coronary Artery Disease in the Japanese
Authors: Zama Takeru , Murata Mitsuru , Matsubara Yumiko , Kawano Koichi , Aoki Nobuo , Yoshino Hideaki , Watanabe Gentaro , Ishikawa Kyozo , Ikeda Yasuo
Published: June 2011

Abstract
Recent reports have suggested that polymorphisms in the human paraoxonase
(HUMPONA)
gene may be a genetic risk factor for coronary artery disease (CAD) in white populations. However, this association has not yet been confirmed in other ethnic populations. We studied 75 Japanese patients with CAD, whose coronary lesions were confirmed by angiography, and 115 Japanese control subjects with no history of CAD and a normal resting electrocardiogram. The assays for genotyping the two polymorphisms in the
HUMPONA
gene (
192
Arg/Gln and
55
Leu/Met) were based on changes in restriction enzyme digestion patterns. For codon 192, the frequencies of the Arg-coding allele (
B
allele) in both patients and control subjects were much higher than those from published results of whites (.26 to .31), and the difference between patients (.74) and control subjects (.59) was statistically significant (
P
=.002). The patient group had a higher proportion of Arg/Arg
(B/B)
homozygotes (52.0% vs 32.2%,
P
=.006). For codon 55, the frequencies of the Leu-coding allele in control subjects and patients were much higher (.91 and .93, respectively) than those published results for whites, but there was no difference between Japanese control subjects and Japanese patients. When subjects with the
55
Leu/Leu genotype only were analyzed,
192
Arg/Arg homozygotes were still significantly more frequent in the patients than in the control subjects (55.4% vs 37.2%,
P
=.024), and the frequency of the
192
Arg allele was also higher in patients than control subjects (
P
=.013). Logistic regression analysis including conventional coronary risk factors revealed that
192
Arg is an independent risk factor for CAD. Thus, in the Japanese, the association of CAD with the
192
Arg variant of
HUMPONA
(B-type enzyme) is similar to that reported for whites, although the allele frequencies for
192
Arg and
55
Leu are much higher in the former than the latter population.
Reactive Oxygen Species Are Involved in Shear Stress-Induced Intercellular Adhesion Molecule-1 Expression in Endothelial Cells
Authors: Chiu J.J. , Wung B.S. , Shyy John Y.J. , Hsieh H.J. , Wang D.L.
Published: June 2011

AbstractVascular endothelial cells (ECs) are constantly subjected to flow-induced shear stress. Although the effects of shear stress on ECs are well known, the intracellular signal mechanisms remain largely unclear. Reactive oxygen species (ROS) have recently been suggested to act as intracellular second messengers. The potential role of ROS in shear-induced gene expression was examined in the present study by subjecting ECs to a shear force using a parallel-plate flow chamber system. ECs under shear flow increased their intracellular ROS as indicated by superoxide production. This superoxide production was maintained at an elevated level as shear flow remained. Sheared ECs, similar to TNFα-, PMA-, or H2O2-treated cells, increased their intercellular adhesion molecule-1 (ICAM-1) mRNA levels in a time-dependent manner. Pretreatment of ECs with an antioxidant,N-acetyl-cysteine (NAC) or catalase, inhibited this shear-induced or oxidant-induced ICAM-1 expression. ROS that were involved in the shear-induced ICAM-1 gene expression were further substantiated by functional analysis using a chimera containing the ICAM-1 promoter region (−850 bp) and the reporter gene luciferase. Shear-induced promoter activities were attenuated by pretreating sheared ECs with NAC and catalase. Flow cytometric analysis and monocytic adhesion assay confirmed the inhibitory effect of NAC and catalase on the shear-induced ICAM-1 expression on ECs. These results clearly demonstrate that shear flow to ECs can induce intracellular ROS generation that may result in an increase of ICAM-1 mRNA levels via transcriptional events. Our findings thus support the importance of intracellular ROS in modulating hemodynamically induced endothelial responses.
Overexpression of the P2Y
2
Purinoceptor in Intimal Lesions of the Rat Aorta
Authors: Seye Cheikh Ibra , Gadeau Alain-Pierre , Daret Danièle , Dupuch Françoise , Alzieu Philippe , Capron Loïc , Desgranges Claude
Published: June 2011

Abstract
Extracellular nucleotides, particularly ATP, are involved in the modulation of arterial vasomotricity via P2 purinoceptors present on smooth muscle and endothelial cells. These nucleotides could also be implicated in the smooth muscle cell hyperplasia observed in intimal lesions. In this study, we tried to define the potential role of the P2Y
2
(P
2u
) purinoceptor by studying its expression in normal and balloon-injured rat aortas. The cloning of a rat P2Y
2
cDNA from a rat smooth muscle cell cDNA library made it possible to study P2Y
2
expression both by Northern blot and in situ hybridization. Northern blot experiments indicated that P2Y
2
mRNA was present in rat medial aortic smooth muscle and in cultured rat aortic smooth muscle cells. In situ hybridization indicated that P2Y
2
mRNA was present in endothelial cells of the intima and in some smooth muscle cells scattered throughout the media of adult rat aortas, while almost all medial smooth muscle cells of rat embryo aorta expressed this receptor. In contrast with adult aortic media, the majority of neointimal smooth muscle cells found in aortic intimal lesions either 8 or 20 days after balloon injury were positive for P2Y
2
mRNA. Moreover, a subpopulation of neointimal cells localized at the luminal surface could be identified by a higher P2Y
2
expression than the underlying neointimal smooth muscle cells. These data showing a strong expression of the P2Y
2
purinoceptor in the neointima of injured arteries suggest that extracellular nucleotides may be involved, via this receptor, in the intimal hyperplasia and/or chronic constriction observed at the lesion site, and consequently in the restenotic process.
Interactions Between Dietary Fat, Fish, and Fish Oils and Their Effects on Platelet Function in Men at Risk of Cardiovascular Disease
Authors: Mori Trevor A. , Beilin Lawrence J. , Burke Valerie , Morris Jenny , Ritchie Jackie
Published: June 2011

Recent studies have suggested that ω3-fats of marine origin may have a protective role in heart disease. This study aimed to compare the effects of fish or fish oil, in the setting of a high- or low-fat diet, on platelet aggregation and platelet thromboxane in men with increased risk of cardiovascular disease. One hundred twenty men who were nonsmokers, 30 to 60 years old, with mildly elevated blood pressure and cholesterol were randomly allocated to one of five high-fat (40% of daily energy) or two low-fat (30%) groups for 12 weeks. The five high-fat groups took either 6 or 12 fish oil capsules daily; fish; a combination of fish and fish oil; or placebo capsules. The two low-fat groups took either fish or placebo capsules. Fish meals provided 1.3 g of eicosapentaenoic acid daily, equivalent to 6 fish oil capsules, and contained an average of 3.65 g/d of ω3-fatty acids. Multiple regression analysis of the combined groups showed that all groups taking ω3-fatty acids reduced platelet aggregation to both collagen (
P
<.0001) and platelet-activating factor (PAF) (
P
<.05) and platelet thromboxane B
2
responses (
P
<.05) to collagen-induced aggregation. The low-fat diet alone had no effect on PAF-induced platelet aggregation and only a small effect on platelet responses to collagen (
P
<.05). Platelet aggregation responses to PAF were reduced more by fish oil than fish in a high-fat diet, whereas fish had a greater effect when part of a low-fat rather than a high-fat diet. There was no significant difference in collagen-induced platelet aggregation or platelet thromboxane between fish and fish oils on a high or low fat intake. In conjunction with our previous findings of improvements in lipoproteins, blood pressure, and heart rate in this population, these results on platelet function suggest that dietary ω3-fatty acids incorporated into a low- rather than a high-fat diet have a wider spectrum of more favorable effects on cardiovascular risk factors.
Role of Leukocyte-Specific LDL Receptors on Plasma Lipoprotein Cholesterol and Atherosclerosis in Mice
Authors: Boisvert William A. , Spangenberg Jörg , Curtiss Linda K.
Published: June 2011

Bone marrow–derived macrophages and lymphocytes express LDL receptors (LDL-R), which allow these cells to take up cholesterol-rich lipoproteins. Although these cells are ubiquitously distributed in the body, it is not known whether they influence plasma cholesterol. Macrophages and T lymphocytes also are found in atherosclerotic lesions, but it is not known whether their LDL-R expression plays a role in atherosclerosis. To address these questions, we subjected LDL-R −/− mice to total body irradiation to eliminate their endogenous bone marrow–derived cells and repopulated them with either LDL-R–expressing wild-type bone marrow (treated mice) or LDL-R −/− bone marrow (control mice). Thus, the only difference between the two groups of mice was the ability of the bone marrow–derived cells to express the LDL-R in the treated mice. Plasma cholesterol levels were similar in the two groups of mice at 8 and 16 weeks after transplantation. Chromatographic separation of the lipoproteins revealed similar lipoprotein cholesterol distributions. Although the extent of lesion area in the aortic valves of the high-fat-diet–fed mice was more severe than that in the chow-fed mice, lesions appeared similar between control and treated mice given either chow or high-fat diet. Abundant LDL-R expression was detected in the lesions of treated mice, whereas the lesions of control mice showed no LDL-R expression, indicating that donor-derived leukocytes had migrated into the lesions of the recipient mice. Thus, bone marrow transplantation can be used as a tool to replace the endogenous bone marrow–derived cells in the artery wall with those of the donor origin.
Basic Fibroblast Growth Factor–Induced Angiogenic Phenotype in Mouse Endothelium
Authors: Bastaki Maria , Nelli Enrico Emanuele , Dell’Era Patrizia , Rusnati Marco , Molinari-Tosatti Maria Pia , Parolini Silvia , Auerbach Robert , Ruco Luigi P. , Possati Laura , Presta Marco
Published: June 2011

Abstract
The mouse is the most commonly used species for in vivo studies on angiogenesis related to tumor development. Yet, to the best of our knowledge, very few reports on the in vitro interaction of the angiogenic basic fibroblast growth factor (bFGF) with mouse endothelial cells are available. Three mouse endothelial cell lines originated from aorta (MAECs), brain capillaries (MBECs), and heart capillaries (MHECs) were characterized for endothelial phenotypic markers, in vivo tumorigenic activity, and the capacity to respond in vitro to bFGF. These cells express angiotensin-converting enzyme, acetylated LDL receptor, constitutive endothelial nitric oxide synthase, and vascular cell adhesion molecule-1 and bind
Griffonia simplicifolia
-I lectin. When injected subcutaneously in nude mice, MAECs induced the appearance of slow-growing vascular lesions reminiscent of epithelioid hemangioendothelioma, whereas MBEC xenografts grew rapidly, showing Kaposi’s sarcoma–like morphological features. No lesions were induced by injection of MHECs. MAECs, MBECs, and MHECs expressed both low-affinity heparan sulfate bFGF-binding sites and high-affinity tyrosine kinase receptors (FGFRs) on their surfaces. In particular, MAECs expressed FGFR-2/
bek
mRNA, whereas microvascular MBECs and MHECs expressed FGFR-1/
flg
mRNA. Accordingly, bFGF induced a mitogenic response and the phosphorylation of extracellular signal-regulated kinase-2 in all the cell lines. In contrast, upregulation of urokinase-type plasminogen activator expression was observed in bFGF–treated microvascular MBECs and MHECs but not in MAECs. Also, bFGF–treated MBECs and MHECs but not MAECs invaded a three-dimensional fibrin gel and formed hollow, capillary-like structures. The relevance of the modifications of the fibrinolytic balance of mouse microvascular endothelium in bFGF–induced angiogenesis was validated in vivo by a gelatin-sponge assay in which the plasmin inhibitors tranexamic acid and ε-aminocaproic acid given to mice in the drinking water inhibited neovascularization induced by the growth factor. In conclusion, differences in response to bFGF exist between large-vessel MAECs and microvascular MBECs and MHECs. Both in vitro and in vivo data point to a role of the profibrinolytic phenotype induced by bFGF in microvascular endothelial cells during mouse angiogenesis. Our observations make these endothelial cell lines suitable for further studies on mouse endothelium during angiogenesis and in angioproliferative diseases.
Notoginsenoside R1 Counteracts Endotoxin-Induced Activation of Endothelial Cells In Vitro and Endotoxin-Induced Lethality in Mice In Vivo
Authors: Zhang Wei-Jian , Wojta Johann , Binder Bernd R.
Published: June 2011

Abstract
In this study we investigated a possible counteracting activity of notoginsenoside R1 (NG-R1) on lipopolysaccharide (LPS)-induced effects in vitro and in vivo. The upregulation of plasminogen activator inhibitor-1 (PAI-1) antigen due to LPS (1 μg/mL for 12 hours) in human umbilical vein endothelial cells (HUVECs) was prevented when the cells were incubated simultaneously with 100 μg/mL NG-R1 (PAI-1 antigen: LPS-treated cells, 969±54 ng/10
5
cells; control cells, 370±15 ng/10
5
cells; LPS+NG-R1–treated cells, 469±29 ng/10
5
cells; n=6). The 2.5- and 3.4-fold (2.2- and 3.2-kb) increases in PAI-1 mRNA levels induced by LPS (1 μg/mL for 6 hours) were reduced to 1.4- and 2.6-fold increases in the presence of both LPS and 100 μg/mL NG-R1. LPS-induced tissue factor (TF) activity in HUVECs was also counteracted when the cells were coincubated with both LPS and 100 μg/mL NG-R1 for 6 hours (TF activity: LPS-treated cells, 88.6±6.5 mU/10
6
cells; control cells, 0.7±0.01 mU/10
6
cells; LPS+NG-R1–treated cells, 56.0±1.9 mU/10
6
cells). The 26-fold increase in TF mRNA levels induced by LPS (1 μg/mL for 2 hours) was reduced to a 13-fold increase in the presence of both LPS and 100 μg/mL NG-R1. PAI activity levels in the plasma of mice 4 hours after injection of LPS (10 ng/g body wt) increased 2.3-fold compared with a control group. In contrast, PAI activity from LPS+NG-R1 (1 μg/g body wt NG-R1)–treated animals was at control level (PAI-1 activity: LPS-treated group, 11.3±3.1 U/mL; control group, 4.9±0.3 U/mL; LPS+NG-R1–treated group, 4.3±1.0 U/mL; n=5 to 8). The production of TNF-α induced by 1 μg/mL LPS by cultured human whole-blood cells was inhibited by 46% when the cells were incubated together with 100 μg/mL NG-R1. NG-R1 protected mice from the lethal effects of LPS. The 78% lethality induced by LPS/galactosamine was reduced to 23% when NG-R1 was administered simultaneously (
P
<.01 by χ
2
test). To extend this study to inflammatory cells, the effect of NG-R1 on LPS stimulation of the monocytic cell line THP-1 was investigated. NG-R1 inhibited the LPS-induced degradation of IκB-α and superinduced LPS-induced IκB-α mRNA, indicating that the effect of NG-R1 is not restricted to endothelial cells and is at least in part mediated by interference with the NF-κB/IκB-α pathway.
Water Diffusion Properties of Human Atherosclerosis and Thrombosis Measured by Pulse Field Gradient Nuclear Magnetic Resonance
Authors: Toussaint Jean-François , Southern James F. , Fuster Valentin , Kantor Howard L.
Published: June 2011

Abstract
Using pulsed field gradient methods combined with magnetic resonance imaging, we calculated the apparent water diffusion coefficient
D
in different atherosclerotic components to probe the microstructure of normal and diseased arteries by characterizing molecular motion.
D
was equal to 0.26±0.13×10
−5
cm
2
·s
−1
in plaque lipid core, 1.45±0.41×10
−5
cm
2
·s
−1
in collagenous cap, and 1.54±0.30×10
−5
cm
2
·s
−1
in normal media. Water diffuses isotropically in the atheromatous core of the plaque, suggesting the absence or destruction of confining structures. The comparable diffusion coefficients in collagenous cap and normal media are consistent with similar biophysical barriers in both components. In thrombi,
D
varies with the aging processes (fresh thrombus, 0.72±0.11×10
−5
cm
2
·s
−1
; 1-week-old thrombus, 0.36±0.08×10
−5
cm
2
·s
−1
; old occluding thrombus, 1.33±0.33×10
−5
cm
2
·s
−1
), consistent with the cross-linking of the fibrin strands occurring in the early phase and the later thrombus organization. Defining an indirect index of arterial lipid infiltration, remodeling, and aging, diffusion imaging provides a new nuclear magnetic resonance characterization of atherothrombosis.
Detection of Osteopontin in Calcified Human Aortic Valves
Authors: Mohler Emile R. , Adam Leonard P. , McClelland Pam , Graham Lori , Hathaway David R.
Published: June 2011

AbstractCardiac valve calcification often results in obstruction of blood flow, which eventually leads to valve replacement. The molecular mechanisms resulting in valve calcification are unknown. Collagen and specific bone matrix proteins are thought to provide the framework for ectopic tissue calcification. This investigation was performed to determine whether the bone matrix protein osteopontin was present in calcified human aortic valves. Proteins extracted from human aortic valve tissue were subjected to polyacrylamide gel electrophoresis followed by Western blotting, using polyclonal antibodies directed against osteopontin. Fresh frozen tissue sections were also screened for osteopontin and macrophages using immunohistochemical techniques. Osteopontin was present in both heavily and minimally calcified aortic valves and absent in noncalcified purely regurgitant or normal aortic valves by both radioimmunoassay (n=16) and immunohistochemical techniques (n=8). Osteopontin colocalized with valvular calcific deposits, and macrophages were identified in the vicinity of osteopontin. These results, in addition to showing that osteopontin is present in calcified human aortic valves, suggest that osteopontin is a regulatory protein in pathological calcification. Identification of the cells producing osteopontin in abnormal cardiac valves and of proximate stimuli for its secretion may lead to novel therapeutic strategies to prevent and/or reverse calcific valve disease.
Fibrin(ogen) and von Willebrand Factor Deposition Are Associated With Intimal Thickening After Balloon Angioplasty of the Rabbit Carotid Artery
Authors: Bosmans J. M. , Kockx M. M. , Vrints C. J. , Bult H. , De Meyer G. R. Y. , Herman A. G.
Published: June 2011

Abstract
The aim of the study was to assess the contribution of thrombus incorporation into neointimal thickening in the rabbit carotid artery after deep vascular injury induced by balloon angioplasty compared with superficial vascular injury induced by a perivascular collar. Besides CD 31 (PECAM I), vimentin, α-smooth muscle actin, rabbit anti-macrophage monoclonal antibody and proliferating cell nuclear antigen, fibrin(ogen) and von Willebrand factor (vWF) deposition was assessed immunohistochemically. Angioplasty was performed in 47 rabbits and evaluated immediately (n=7), after 6 hours (n=4), and after 1 (n=7), 2 (n=9), or 3 (n=20) weeks. A collar was placed in 29 rabbits and evaluated immediately (n=5), after 6 hours (n=5), and after 1 (n=7), 2 (n=10), or 3 (n=2) weeks. After dilatation, the arteries were extensively denuded of endothelium, the internal elastic membrane was ruptured and blood-filled clefts were present in the media, pointing to deep vascular (type III) injury. Six hours later, mural fibrin(ogen) thrombi were formed, specially at sites with severe damage. This fibrin(ogen) matrix became infiltrated by phagocytes and smooth muscle cells. A luminal cap covered by regenerating endothelium was formed, demonstrating increased immunoreactivity to vWF. vWF was deposited in the extracellular neointimal spaces. Fibrin(ogen) thrombus deposition and incorporation appeared to be protracted phenomena for at least 2 weeks. After collar placement, minimal endothelial denudation was documented, pointing to focal superficial (type I) vascular injury. In subsequent weeks, neointimal thickening was associated with vWF deposition but not with fibrin(ogen) thrombus incorporation. In conclusion, mural fibrin(ogen) thrombus formation and incorporation contribute to neointima formation after deep vascular injury and seem to occur for several weeks after the initial insult.
Cellular Dynamics in Early Atherosclerotic Lesion Progression in White Carneau Pigeons
Authors: Jerome W. Gray , Lewis Jon C.
Published: June 2011

Abstract
The early stages of atherosclerosis are characterized by macrophage invasion of the intimal space and proliferation of smooth muscle cells (SMCs). In this study, we characterize the timing and location of each of these processes. Dividing cells of the developing lesions were labeled with [
3
H]thymidine at different times during lesion progression. Comparable stages of lesion development were analyzed, with the only variable being the length of time between labeling and necropsy. In this way, the mechanism by which lesion composition changes could be inferred from changes in the distribution of label over time. When cells were labeled just before necropsy, serial sections showed monocyte influx predominantly occurring at lesion edges and resulting in a labeling index of 27%. Near the lesion edge, medial SMCs were not stimulated to proliferate. In contrast, in more central areas of the lesion, monocyte influx and intimal SMC proliferation were minimal. Medial SMC proliferation in central regions, however, was high, with a labeling index of 20% in the upper media. Over time, proliferating medial SMCs migrated into the intimal lesion but did not migrate laterally to any great extent. This migration pattern maintained precise subdomains during lesion development, with each domain representing a different stage of lesion development. The edge of lesions thus represents very early events, central regions indicate later events, and intermediate regions provide information about stages between these extremes.
N-α-Tosyl-l-Lysine Chloromethylketone Prevents Expression of iNOS in Vascular Smooth Muscle by Blocking Activation of NF-κB
Authors: Schini-Kerth Valérie B. , Boese Matthias , Busse Rudi , Fisslthaler Beate , Mülsch Alexander
Published: June 2011

AbstractCertain cytokines and lipopolysaccharide stimulate expression of inducible nitric oxide synthase (iNOS) in vascular smooth muscle, an event that is regulated at the transcriptional level and appears to involve several transcription factors, including nuclear factor κB (NF-κB). Since proteases play an essential role in NF-κB activation, experiments were designed to clarify, in both cultured rat aortic smooth muscle cells (SMCs) and isolated rat aortas, whether protease inhibitors affect the interleukin-1β (IL-1β)–elicited expression of iNOS. The formation of NO was assessed by nitrite release in cultured SMCs and the attenuation of phenylephrine-induced contraction in aortic rings, the expression of iNOS by Western blot analysis and reverse transcription–polymerase chain reaction, and NF-κB activity in nuclear extracts by gel electrophoretic mobility shift assay. Exposure of cultured SMCs to IL-1β increased NF-κB binding activity within 30 minutes and was associated with nitrite accumulation and the appearance of iNOS protein 24 hours later. These responses were abolished in cells that had been exposed to the cytokine in the presence of the protease inhibitorN-α-tosyl-l-lysine chloromethylketone. Aprotinin andp-toluenesulfonyl-l-arginine methyl ester, two other protease inhibitors, also reduced the cytokine-stimulated release of nitrite and the level of iNOS protein. Exposure of rat aortic segments without endothelium to IL-1β activated NF-κB within 30 minutes and was associated with the appearance of iNOS mRNA and an attenuation of phenylephrine-induced contraction 6 hours later. These responses were blunted when the segments were incubated with the cytokine andN-α-tosyl-l-lysine chloromethyl ketone. The present observations indicate that protease inhibitors prevent iNOS expression in both cultured and native vascular SMCs by blocking the activation of NF-κB.
Lipid Oxidation Products Have Opposite Effects on Calcifying Vascular Cell and Bone Cell Differentiation
Authors: Parhami Farhad , Morrow Andrew D. , Balucan Jennifer , Leitinger Norbert , Watson Andrew D. , Tintut Yin , Berliner Judith A. , Demer Linda L.
Published: June 2011

Abstract
Atherosclerotic calcification and osteoporosis often coexist in patients, yielding formation of bone mineral in vascular walls and its simultaneous loss from bone. To assess the potential role of lipoproteins in both processes, we examined the effects of minimally oxidized low-density lipoprotein (MM-LDL) and several other lipid oxidation products on calcifying vascular cells (CVCs) and bone-derived preosteoblasts MC3T3-E1. In CVCs, MM-LDL but not native LDL inhibited proliferation, caused a dose-dependent increase in alkaline phosphatase activity, which is a marker of osteoblastic differentiation, and induced the formation of extensive areas of calcification. Similar to MM-LDL, oxidized 1-palmitoyl-2-arachidonoyl-
sn
-glycero-3-phosphorylcholine (ox-PAPC) and the isoprostane 8-iso prostaglandin E
2
but not PAPC or isoprostane 8-iso prostaglandin F
2α
induced alkaline phosphatase activity and differentiation of CVCs. In contrast, MM-LDL and the above oxidized lipids inhibited differentiation of the MC3T3-E1 bone cells, as evidenced by their stimulatory effect on proliferation and their inhibitory effect on the induction of alkaline phosphatase and calcium uptake. These results suggest that specific oxidized lipids may be the common factors underlying the pathogenesis of both atherosclerotic calcification and osteoporosis.
Phenotypic Variation in Patients Heterozygous for Familial Defective Apolipoprotein B (FDB) in Three European Countries
Authors: Hansen P.S. , Defesche J.C. , Kastelein J.J.P. , Gerdes L.U. , Fraza L. , Gerdes C. , Tato F. , Jensen H.K. , Jensen L.G. , Klausen I.C. , Faergeman O. , Schuster H.
Published: June 2011

Abstract
A glutamine-for-arginine substitution at amino acid position 3500 of apolipoprotein B (apo B) causes synthesis of LDL with reduced binding affinity to the LDL receptor (LDLR). The associated clinical syndrome has been named familial defective apolipoprotein B-100 (FDB). In 205 FDB patients from Germany (n=73), the Netherlands (n=87), and Denmark (n=45), we tried to assess determinants of variation in lipid concentrations. Besides age, sex, and geographic origin, variation in the LDLR gene was the most powerful determinant of variation in total cholesterol and LDL cholesterol levels. Polymorphic variation in the LDLR gene (
Sfa
NI, exon 2;
Nco
I, exon 18) was associated with total cholesterol (TC) and LDL cholesterol (LDL-C) variation in women (
Sfa
NI:
P
=.04 and .03 for TC and LDL-C, respectively;
Nco
I:
P
=.003 and .006, respectively), whereas the
Ava
II (exon 13) and the
Pvu
II (intron 15) polymorphisms were not. Combined information from all three LDLR exon polymorphisms showed that subjects with at least one S+A+N+ allele had 13% to 20% higher TC than non-S+A+N+ subjects (
P
=.02 [TC, men];
P
=.01 [LDL-C, men];
P
=.005 [TC, women]; and
P
=.004 [LDL-C, women]) and, together with age and geographic origin, accounted for 20% (women) and 19% (men) of the variation in LDL-C. The expected association of the apo E genotypes (e
3
e
2
, e
3
e
3
, and e
3
e
4
) with cholesterol concentrations was seen in S+A+N+ but not in non-S+A+N+ subjects and in P−P− but not in P+P+ or P+P− subjects. With regard to clinical expression, FDB patients had lower TC and LDL-C levels and a lower prevalence of cardiovascular disease than 101 Danish patients with familial hypercholesterolemia.