bluuebunny/crossref_metadata_2025 huggingface dataset:
Shear-Induced Platelet Activation and Platelet Microparticle Formation at Blood Flow Conditions as in Arteries With a Severe Stenosis
Authors: Holme Pål A. , Ørvim Una , Hamers Maria J. A. G. , Solum Nils O. , Brosstad Frank R. , Barstad R. Marius , Sakariassen Kjell S.
Published: June 2011

Abstract
In the present study, we investigated whether high arterial shear stresses at various exposure times or a sudden increase in shear stress introduced by a stenosis affect platelet activation and platelet microparticle formation in native human blood. We used a parallel-plate perfusion chamber device through which nonanticoagulated human blood was drawn (10 mL/min) by a pump directly from an antecubital vein through the flow channel of a perfusion chamber at wall shear rates of 420, 2600, and 10 500 s
−1
. In another set of experiments, an eccentric stenosis was introduced into the flow channel. Wall shear rates of 2600 or 10 500 s
−1
at the stenosis apex were maintained at the same flow rate. The wall shear rate upstream and downstream of these stenoses was 420 s
−1
. A shear rate of 420 s
−1
is within the range of those encountered in healthy small coronary arteries, whereas those of 2600 and 10 500 s
−1
are representative for vessels with various degrees of stenotic lesions. The blood was exposed to these shear rates for periods varying from 0.075 to 3.045 seconds. Platelet activation was assessed as activated glycoprotein (GP) IIb/IIIa by FITC-labeled monoclonal antibody (MAb) PAC-1 and aminophospholipid translocation by FITC-labeled annexin V. Microparticle formation was quantified by FITC-labeled MAb Y2/51 directed against GP IIIa. Significant platelet activation and formation of microparticles were observed at 10 500 s
−1
only (
P
<.008). This shear-induced platelet activation and microparticle formation were enhanced by introduction of a thrombus-promoting surface consisting of type III human collagen fibrils. Introduction of the most severe stenosis at 10 500 s
−1
further increased platelet activation (
P
<.017). The collagen-induced thrombus formation increased the platelet thrombus volume at 10 500 s
−1
from 16.5 to 33.8 μm
3
/μm
2
(
P
<.003) on the stenosis apex when the most severe stenosis was used. A correlation (
P
<.0001) between platelet thrombus volume and platelet microparticle formation was observed in the presence of the eccentric stenoses. Apparently, high shear stress (315 dynes/cm
2
at 10 500 s
−1
), as encountered in severe atherosclerotic arteries, activated platelets and triggered platelet microparticle formation. In contrast, no significant platelet activation or formation of platelet microparticles was observed at physiological shear (420 s
−1
) or at the shear condition simulating shear in arteries with a less severe stenosis (2600 s
−1
). The data imply that platelets are activated and form microparticles in native blood at very high shear stresses. These events are potentiated by prolonged exposure to the high shear or by a sudden change of increasing shear due to the stenosis. The latter situation apparently enhances platelet thrombus formation at the stenosis.
Differentiation, Dedifferentiation, and Apoptosis of Smooth Muscle Cells During the Development of the Human Ductus Arteriosus
Authors: Slomp Jennichjen , Gittenberger-de Groot Adriana C. , Glukhova Marina A. , van Munsteren J. Conny , Kockx Mark M. , Schwartz Stephen M. , Koteliansky Victor E.
Published: June 2011

Abstract
Differentiation of vascular smooth muscle cells (SMCs) is characterized by several molecular transitions. As differentiation proceeds, proteins of the cytoskeletal and contractile apparatus, such as α-smooth muscle actin, smooth muscle myosin, calponin, and heavy caldesmon, and the expression of the membrane-related protein smooth muscle phosphoglucomutase–related protein increase, whereas the expression of other proteins, such as fibronectin splice variants with extradomains A (EDA) and B (EDB), decreases. In this study, we investigated the differentiation of the SMCs of the ductus arteriosus during the development of intimal thickening. Ascending and descending aortas of the same age were used for comparison because these vessels lack intimal thickening. In the fetal ductus arteriosus, a relatively early differentiation of the contractile apparatus was observed compared with the ascending and descending aortas. EDA and EDB expression was already low, being similar in the ductus and descending aorta and even lower in the ascending aorta. In the neonatal ductus, SMCs of the media and outer intima were well differentiated and comparable with SMCs of the ascending aorta. Dedifferentiated SMCs, with a low expression of cytoskeletal and contractile proteins and a high expression of EDA and EDB, were found in regions in the inner intima that show features of progression of intimal thickening and in areas of cytolytic necrosis in the media. With a technique using in situ end labeling of DNA fragments, we found extensive apoptosis in the area of cytolytic necrosis and to a lesser extent in these areas of the inner intima. In conclusion, SMCs of the fetal ductus arteriosus have an advanced differentiation of the contractile apparatus compared with the adjacent aorta. Reexpression of fetal characteristics is seen in a number of cells in inner intima and media of the neonatal ductus arteriosus. The finding of apoptosis in these areas suggests that dedifferentiation and apoptosis are associated processes that may play a role in vascular remodeling.
No Evidence of Linkage Between Familial Combined Hyperlipidemia and Genes Encoding Lipolytic Enzymes in Finnish Families
Authors: Pajukanta Päivi , Porkka Kimmo V.K. , Antikainen Marjatta , Taskinen Marja-Riitta , Perola Markus , Murtomäki-Repo Sanna , Ehnholm Sonja , Nuotio Ilpo , Suurinkeroinen Leena , Lahdenkari Anne-Tiina , Syvänen Ann-Christine , Viikari Jorma S.A. , Ehnholm Christian , Peltonen Leena
Published: June 2011

Abstract
Familial combined hyperlipidemia (FCHL) is characterized by different lipid phenotypes (IIa, IIb, IV) and elevated apolipoprotein B (apo B) levels in affected family members. Despite intensive research, the genes involved in the expression of this complex disorder have not been identified, probably because of problems associated with phenotype definition, unknown mode of inheritance, and most probably genetic heterogeneity. To explore the genetics of FCHL in the genetically homogeneous Finnish population, we collected 14 well-documented Finnish pedigrees with premature coronary heart disease and FCHL-like dyslipidemia. The lipolytic enzymes lipoprotein lipase (LPL), hepatic lipase (HL), and hormone-sensitive lipase (HSL) were selected as initial candidate genes because of their central roles in apo B and triglyceride metabolism. On the basis of the pedigree structures, a dominant mode of inheritance was adopted for linkage analyses, and serum total cholesterol and/or triglyceride levels exceeding the 90th percentile level were set as diagnostic criteria (criterion 1). In pairwise linkage analyses with intragenic markers, no evidence for linkage was found. Instead, the significantly negative LOD scores suggested exclusion of all three loci for single major gene effect. LOD scores were −14.63, −5.03, and −5.70 for the three LPL polymorphisms (Θ=0.00); −9.40, −6.30, and −4.74 for the three HL polymorphisms (Θ=0.00); and −15.29 for the HSL polymorphism (Θ=0.00). The results were very similar when apo B levels over the 90th percentile were used as criteria for affected status (criterion 2). Also, when linkage calculations were carried out using an intermediate or recessive mode of inheritance, the results of pairwise linkage analysis remained negative. Furthermore, when haplotypes were constructed from multiple polymorphisms of the LPL and HL genes, no segregation with the FCHL phenotype could be observed in the 14 Finnish families. Data obtained by the affected sib-pair method supported these findings, suggesting that the LPL, HL, or HSL genes do not represent major loci influencing the expression of the FCHL phenotype.
Decreased Production and Increased Catabolism of Apolipoprotein B-100 in Apolipoprotein B-67/B-100 Heterozygotes
Authors: Welty Francine K. , Lichtenstein Alice H. , Barrett P. Hugh R. , Dolnikowski Gregory G. , Ordovas Jose M. , Schaefer Ernst J.
Published: June 2011

Abstract
Apolipoprotein (apo) B-67 is a truncated form of apoB-100 due to deletion of an adenine at cDNA 9327. Heterozygotes have one allele making apoB-100; therefore, plasma apoB levels would be predicted to be at least 50% of normal. However, apoB-67 heterozygotes have total plasma apoB levels that are 24% of normal. To determine the mechanisms responsible for the lower-than-expected levels of apoB, in vivo kinetics of apoB-100 were performed in three apoB-67/apoB-100 heterozygotes and compared with those of six control subjects by using a primed-constant infusion of [5,5,5-
2
H
3
]leucine in the fed state. Kinetic parameters were calculated by multicompartmental modeling of the data. The mean total apoB plasma concentration of the apoB-67 subjects was 21.8±6.1 mg/dL, or 24% of that of control subjects (89.6±24.1 mg/dL,
P
=.002). ApoB-67 subjects had lower mean VLDL apoB-100 production rates (3.6±1.2 versus 13.9±3.5 mg·kg
−1
·d
−1
,
P
=.002) and lower mean transport rates of apoB-100 into LDL (3.5±1.4 versus 12.6±4.1 mg·kg
−1
·d
−1
,
P
=.008) compared with control subjects. The transport rate into IDL was not significantly different (1.2±0.5 versus 6.2±4.0 mg·kg
−1
·d
−1
,
P
=.07). The fractional catabolic rate of VLDL apoB-100 was significantly higher in apoB-67 subjects than in control subjects (18.1±8.6 versus 7.6±1.6 mg·kg
−1
·d
−1
,
P
=.017). ApoB-100 IDL and LDL fractional catabolic rates were not significantly different. VLDL apoB-100 pool size in apoB-67 subjects was 11% of that of control subjects (15.8±7.7 versus 141.6±33.7 mg,
P
=.0004) due to a 74% lower production rate (26% of control values) and a 2.4-fold higher fractional catabolic rate. LDL apoB-100 pool size in apoB-67 subjects was 22% of that of control subjects (665.3±192.4 versus 2968.3±765.2 mg,
P
=.002) due primarily to a lower production rate (27% of control values). Thus, both decreased production of VLDL and LDL apoB-100 and increased catabolism of VLDL apoB-100 are responsible for the low levels of apoB-100 in apoB-67 subjects.
Acetylated LDL Endocytosis by the Human Monocytic Mono Mac 6sr Cells Is Not Mediated by the Macrophage Type I and II Scavenger Receptors
Authors: Scheithe Rupert , Heidenthal Anne K. , Danesch Ulrich , Mauthner Eva , Hapfelmeier Gerhard , Becker Alexander , Pietsch Angelika , Weber Peter C. , Hrboticky Nina
Published: June 2011

AbstractWe recently reported that the human monocytic Mono Mac 6sr cell line constitutively takes up and degrades acetylated (acLDL) and oxidized LDL through receptor-specific pathways. The present studies were undertaken to further characterize the acLDL binding site on a functional and molecular basis. The degradation of acLDL increased during differentiation of Mono Mac 6sr cells with lipopolysaccharide (10 ng/mL, 72 hours) and low concentrations of phorbol 12-myristate 13-acetate (PMA; 0.1 to 1.0 ng/mL, 72 hours). Higher doses of PMA (5 or 10 ng/mL), however, decreased acLDL degradation. Scatchard plots of acLDL binding in untreated and LPS-differentiated Mono Mac 6sr cells were nonlinear and suggested the presence of more than one binding site. Although the ligand specificity of the acLDL receptor in Mono Mac 6sr cells resembles that of the macrophage type I and type II scavenger receptors, we did not detect mRNA of either receptor type in untreated or differentiated Mono Mac 6sr cells by means of Northern blotting and reverse transcription polymerase chain reaction. Furthermore, ligand blotting with125I-acLDL failed to detect the 220-kD types I and II scavenger receptor protein. Thus, Mono Mac 6sr cells express an acLDL receptor that is distinct from the type I and type II scavenger receptor found in human monocyte–derived macrophages but that, like the latter, is induced during monocytic differentiation.
Associations of HDL
2
and HDL
3
Subfractions With Ischemic Heart Disease in Men
Authors: Lamarche Benoît , Moorjani Sital , Cantin Bernard , Dagenais Gilles R. , Lupien Paul J. , Després Jean-Pierre
Published: June 2011

Abstract
Individuals with elevated plasma concentrations of HDL cholesterol are at lower risk for ischemic heart disease (IHD). Whether the cardioprotective effects of HDL can be attributed to one or both HDL subfractions (HDL
2
and HDL
3
) remains, however, controversial. The relationship of HDL subfractions to the incidence of IHD was investigated in a sample of 1169 French-Canadian men younger than 60 years and living in the Quebec City suburbs. Between 1980 to 1981 and 1990, 83 of the 944 men with complete follow-up in 1990 (80.8%) had a first IHD. Men who developed IHD had lower HDL, HDL
2
, and HDL
3
cholesterol concentrations at baseline than men who remained free from IHD. Adjusted relative risk (RR) of IHD was calculated among quartiles of HDL cholesterol and HDL subfractions with the use of Cox survival models. Men in the fourth quartile of HDL
2
(RR=0.21; 95% confidence interval [CI], 0.08 to 0.56) and HDL
3
cholesterol distributions (RR=0.37; 95% CI, 0.15 to 0.94) were at lower risk for IHD than men in the first quartile. Despite the fact that the respective contributions of HDL
2
and HDL
3
to IHD risk were of the same magnitude in a multivariate model that included both subfractions, the contribution of the HDL
2
subfraction was statistically significant (standardized RR=0.84; 95% CI, 0.74 to 0.95), whereas it did not reach significance for HDL
3
(standardized RR=0.87; 95% CI, 0.69 to 1.11). Neither the linear combination of HDL
2
and HDL
3
nor their ratio provided further information on the risk of IHD compared with HDL cholesterol alone or with the ratio of total to HDL cholesterol. >From a statistical standpoint, the present data suggest that the HDL
2
subfraction may be more closely related to the development of IHD than the HDL
3
subfraction. However, the qualitative difference in the relative predictive value of each subfraction was trivial, since it only corresponded to a modest quantitative difference. Thus, the possibility that a significant proportion of the cardioprotective effect of elevated HDL cholesterol levels may be mediated by the HDL
3
subfraction still cannot be excluded. Finally, from a clinical point of view and within the limits of resolution provided by these data, the measurement of HDL subfractions does not appear to provide any additional information on the risk of IHD than HDL cholesterol alone or the ratio of total to HDL cholesterol.
Parathyroid Hormone–Related Peptide as a Local Regulator of Vascular Calcification
Authors: Jono Shuichi , Nishizawa Yoshiki , Shioi Atsushi , Morii Hirotoshi
Published: June 2011

AbstractIn the present study, we investigated the role of parathyroid hormone–related peptide (PTHrP) in vascular calcification by using an in vitro calcification model. We demonstrated that the expression of PTHrP decreased in the progression of bovine vascular smooth muscle cell (BVSMC) calcification and that inhibition of calcification by etidronate (EHDP) and levamisole restored PTHrP secretion, suggesting that the expression of PTHrP is associated with calcification. PTHrP (1-34) and PTH (1-34) dose-dependently inhibited BVSMC calcification. Protein kinase A (PKA) and protein kinase C (PKC) inhibitors completely blocked the inhibitory effect of PTHrP, suggesting that both PKA and PKC may be involved in its signaling pathway. Moreover, PTHrP inhibited alkaline phosphatase (ALP) activity, implying that the impact on ALP may contribute to its action on calcification. Furthermore, the PTHrP antagonist, PTHrP (7-34), dose-dependently increased calcium deposition by BVSMC. Interestingly, PTHrP production by BVSMC dramatically increased in the presence of EHDP, and PTHrP (7-34) partially antagonized the inhibitory effect of EHDP on BVSMC calcification. These results suggest that PTHrP may regulate vascular calcification as an autocrine/paracrine factor.
Prevention of Aortic Fibrosis by Spironolactone in Spontaneously Hypertensive Rats
Authors: Benetos A. , Lacolley P. , Safar M.E.
Published: June 2011

Abstract
We have previously shown that long-term angiotensin-converting enzyme (ACE) inhibition prevents the increase in aortic collagen in spontaneously hypertensive rats (SHRs), independent of blood pressure reduction. More recently, we reported that the effects of ACE inhibition in the prevention of aortic collagen accumulation were related to the inhibition of angiotensin II actions on angiotensin II type 1 receptors. Aldosterone, the synthesis of which is mainly modulated by angiotensin II through type 1 receptor stimulation, is known to promote cardiac fibrosis in different experimental models. The aim of the present study was to determine whether inhibition of aldosterone formation was able to prevent aortic fibrosis in SHRs. For this purpose, we compared the effects of a 4-month treatment with the aldosterone antagonist spironolactone with the ACE inhibitor quinapril in 4-week-old SHRs. Control SHRs and Wistar-Kyoto (WKY) rats received placebo for the same period of time. At the end of treatment, in conscious SHRs vs WKY controls, quinapril completely prevented the development of hypertension, whereas spironolactone produced only a slight but significant reduction in blood pressure. Aortic hypertrophy was significantly prevented by ACE inhibition but not by spironolactone. On the contrary, aortic collagen accumulation was completely prevented by both quinapril and spironolactone. In the latter case, collagen density was significantly below that of WKY controls. These results show that in SHRs, spironolactone can markedly prevent aortic fibrosis in the presence of a very slight antihypertensive effect. It is suggested that ACE inhibition or type 1 receptor antagonist–induced prevention of aortic collagen accumulation is at least partially related to aldosterone inhibition.
Vitamin C Reduces Cholesterol-Induced Microcirculatory Changes in Rabbits
Authors: Freyschuss Anna , Xiu Rui-Juan , Zhang Jian , Ying Xiaoyou , Diczfalusy Ulf , Jogestrand Tomas , Henriksson Peter , Björkhem Ingemar
Published: June 2011

Abstract
The microcirculation was studied for 10 weeks in untreated rabbits (n=12) and in rabbits treated with vitamin C in their drinking water (0.5 g/d; n=6), a 1% cholesterol diet (n=12), or a combination of the two treatments (n=11). The studies were performed by direct intravital microscopic imaging of the conjunctiva of both eyes to evaluate blood flow velocity, microvessel diameter, and microhemorheologic conditions. As we reported previously, changes occurred in all of the aforementioned variables as a consequence of cholesterol feeding. After 3 and 6 weeks of feeding, there was a marked and significant (
P
<.0001) decrease in blood flow velocity in third-order arterioles, which was accompanied by stasis and erythrocyte aggregation in the smaller conjunctival vessels. When cholesterol treatment was combined with vitamin C, blood flow was almost identical to that of controls and significantly (
P
<.0001) higher than that of rabbits treated with cholesterol alone. All other changes were also significantly reduced by the addition of vitamin C treatment to the cholesterol diet. Cholesterol-treated rabbits developed macroscopic arterial lesions that were not significantly reduced by vitamin C treatment. Neither circulating oxysterol levels nor atheromas were reduced by vitamin C treatment, which also had no significant effect on lipid or circulating vitamin E levels. We have previously shown that the lipid-soluble antioxidant BHT is able to prevent both cholesterol-induced microcirculatory changes and the development of arterial lesions in rabbits. This phenomenon is compatible with a critical oxidation step occurring in the lipid phase that is common to both processes. The finding that microcirculatory changes can be prevented by a water-soluble antioxidant is compatible with a role for water-soluble oxidants in this context. The possibility is discussed that vitamin C might also be important for the microcirculation in humans.
Inhibitory Effects of Hypercholesterolemia and Ox-LDL on Angiogenesis-like Endothelial Growth in Rabbit Aortic Explants
Authors: Chen Chu-Huang , Cartwright Joiner , Li Zheng , Lou Sherry , Nguyen Hai Hoang , Gotto Antonio M. , Henry Philip D.
Published: June 2011

Abstract
Hypercholesterolemic (HC) rabbits exhibit suppressed compensatory vascular growth after restriction of arterial supply. However, neovascularization is commonly found in atheromas containing inflammatory cells. We used an in vitro model to determine the effects of hypercholesterolemia on angiogenesis in the absence or presence of inflammatory cells. HC rabbit aortic explants (1 mm
2
) with or without (n=90 each) lesion-forming inflammatory cells were cultured in a collagen matrix with serum-free medium. Explant-derived endothelial cell growth was organized into capillary-like microtubes (CLM) that could be videomicroscopically quantified. CLM growth from lesion-free HC explants was significantly reduced to 13±4% of the value in explants (n=90) from normocholesterolemic (NC, n=15) rabbits (
P
<.001). In contrast, in lesion-containing HC explants, the matrix was invaded by foam cells, and CLM growth was not inhibited. Immunoassayable basic fibroblast growth factor (bFGF, in pg/mL) in the culture medium was significantly lower in lesion-free HC (<5) than NC explants (11±2,
P
<.01) or HC explants with lesions (14±3). In addition, CLM growth was reduced in NC explants incubated with oxidized LDL (ox-LDL, 50-100 μg/mL). Exogenous bFGF (10 ng/mL) reversed the inhibitory effects of hypercholesterolemia and ox-LDL, whereas bFGF-neutralizing antibody (10 μg/mL) abolished CLM growth in all groups. In cultured rabbit aortic endothelial cells, ox-LDL reduced DNA synthesis, but this inhibition was reversed by bFGF. We conclude that hypercholesterolemia and ox-LDL inhibit angiogenesis-like endothelial growth because of a suppressed availability of endogenous bFGF. Retained responsiveness to exogenous bFGF suggests that inducing bFGF expression at targeted sites may improve collateral growth in hyperlipidemic arterial disease.
Modulation of Tissue Factor Protein Expression in Experimental Venous Bypass Grafts
Authors: Channon Keith M. , Fulton Gregory J. , Davies Mark G. , Peters Kevin G. , Ezekowitz Michael D. , Hagen Per-Otto , Annex Brian H.
Published: June 2011

AbstractVein graft failure is a major limitation of coronary artery and peripheral vascular surgery. Tissue factor (TF), a transmembrane glycoprotein, generates thrombin by initiating the extrinsic coagulation cascade and plays a major role in the response to arterial injury. This study was designed to examine changes in TF protein expression in response to venous bypass grafting. New Zealand White rabbits underwent interposition bypass grafting of the common carotid artery via the ipsilateral external jugular vein. The contralateral control jugular veins (n=6), early vein grafts (1 or 3 days after grafting, n=18), and late vein grafts (14 or 28 days after grafting, n=8) were examined by immunohistochemistry. The presence or absence of TF immunostaining in the intima was assessed in each vessel quadrant. In control veins, intimal TF staining was present in 5 of 24 vessel quadrants. In early vein grafts, TF staining was markedly increased in the intima (72 of 72 quadrants,P<.001 vs control veins), and TF immunostaining colocalized with CD18-positive leukocytes but not with endothelial cells, vascular smooth muscle cells, or RAM11-positive macrophages. In late vein grafts with intimal hyperplasia, TF expression was low or absent in the intima (6 of 32 quadrants,P<.001 vs early vein grafts;P=NS vs control veins), although medial smooth muscle cells expressed TF. Marked changes in TF expression occur in vein grafts. In early vein grafts, TF protein was greatly increased in the intima for at least 3 days and was associated with CD18-positive leukocytes. In late vein grafts with intimal hyperplasia, however, TF protein was not seen in the intima. These findings may have important implications for the development of therapeutic strategies to limit vein graft failure.
Platelet Activation in Flowing Blood Passing Growing Arterial Thrombi
Authors: Hagberg Inger Anne , Roald Helge Einar , Lyberg Torstein
Published: June 2011

Abstract
We investigated the combined effect of wall shear rate and immobilized collagen on platelet activation in flowing nonanticoagulated human blood. By combining an ex vivo model of thrombogenesis with flow cytometry, we showed that activated platelets can be detected in the bloodstream passing growing thrombi at a wall shear rate characteristic of moderately stenosed arteries (2600 s
−1
). The activation of the circulating platelets was clearly correlated with thrombus growth. Different antibodies against platelet activation-dependent surface markers had distinct sensitivity to the thrombotic process. α-Granule release detected by surface expression of CD62P seemed to be the most sensitive marker, as judged by both mean fluorescence intensity and fraction of platelets activated. The conformational change in glycoprotein IIb–IIIa, as detected by PAC-1, also seemed to be a sensitive marker and preceded binding of fibrinogen to activated glycoprotein IIb–IIIa, as detected by anti-fibrinogen. Large thrombi also elicited lysosome exocytosis, detected by surface expression of CD63. Finally, we observed a small decrease of glycoprotein Ib–IX expression, as detected by anti-CD42a. Thus, our study provides further information on the dynamics of platelet activation in relation to thrombus growth at arterial shear conditions in flowing nonanticoagulated human blood.
Familial Moderate Hypercholesterolemia Caused by Asp235→Glu Mutation of the LDL Receptor Gene and Co-occurrence of a De Novo Deletion of the LDL Receptor Gene in the Same Family
Authors: Koivisto Ulla-Maija , Gylling Helena , Miettinen Tatu A. , Kontula Kimmo
Published: June 2011

AbstractWe identified a large family in which a hitherto unreported point mutation of the LDL receptor gene (Asp235→Glu) cosegregated with moderately elevated serum LDL cholesterol concentration. Within one generation, the mean serum total and LDL cholesterol levels in four heterozygous carriers of this mutation (7.76±1.46 and 5.89±1.56 mmol/L, respectively) were significantly (P<.05) higher than the corresponding concentrations in their five nonaffected siblings (5.81±0.57 and 3.77±0.54 mmol/L, respectively). Lipid levels in carriers of the Asp235→Glu mutation were, however, markedly lower than the corresponding total and LDL cholesterol levels (about 12 and 10 mmol/L, respectively) in heterozygous patients with the two common LDL receptor mutations (FH-Helsinki and FH-North Karelia). None of the four siblings in the age range of 54 to 69 years had experienced a myocardial infarction, although symptoms suggestive of coronary artery disease were present in two and tendon xanthomas were found in one. Expression of the mutant receptor in COS cells indicated an approximately 50% to 70% reduction of LDL-binding activity compared with the normal receptor. One patient (female, aged 39 years) had severe hypercholesterolemia in the range of 13 to 20 mmol/L when untreated, extensive coronary artery disease as demonstrated by angiography, and extensor tendon xanthomatosis. In addition to the Asp235→Glu mutation, she was found to have a de novo deletion of exons 14 and 15 in her other LDL receptor allele. In this subject, the total LDL receptor activity of mitogen-stimulated blood lymphocytes was very low. In conclusion, along with another LDL receptor gene mutation (FH-Espoo or deletion of exon 15) described by us previously, the Asp235→Glu mutation (designated as FH-Keuruu) indicates that moderate varieties of inherited hypercholesterolemia may result from LDL receptor gene mutations of mild expression.
Lysophosphatidylcholine Inhibits Receptor-Mediated Ca
2+
Mobilization in Intact Endothelial Cells of Rabbit Aorta
Authors: Miwa Yoichi , Hirata Ken-ichi , Kawashima Seinosuke , Akita Hozuka , Yokoyama Mitsuhiro
Published: June 2011

Abstract
We have previously reported that lysophosphatidylcholine (LPC), which accumulates in oxidized LDL and atherosclerotic arteries, inhibits endothelium-dependent relaxation and modulates Ca
2+
regulation in cultured bovine aortic endothelial cells. To test the effect of LPC on endothelium-dependent relaxation and endothelial Ca
2+
regulation in intact vessels, we simultaneously measured both isometric tension and endothelial cytosolic free Ca
2+
concentration ([Ca
2+
]
i
), using fura 2, in intact endothelial cells of aortic strips isolated from rabbits. In the aortic strips precontracted with phenylephrine, cumulative addition of acetylcholine (ACh) dose dependently induced endothelium-dependent relaxation, with an increase in endothelial [Ca
2+
]
i
, and positive correlation was obtained between these two parameters. LPC (2 to 20 μmol/L) inhibited both ACh (3 μmol/L)-induced endothelium-dependent relaxation and an increase in endothelial [Ca
2+
]
i
in a dose-dependent manner. On the other hand, phosphatidylcholine (20 μmol/L) affected neither ACh-induced endothelium-dependent relaxation nor an increase in endothelial [Ca
2+
]
i
. LPC had no effect on endothelium-independent relaxation and a decrease in smooth muscle [Ca
2+
]
i
induced by nitroglycerin. Thus, the inhibitory effect of LPC on endothelium-dependent relaxation is due to the inhibition of agonist-induced Ca
2+
mobilization in vascular endothelial cells, which is an essential step in the synthesis of endothelium-derived relaxing factor.
Role of Free Apolipoprotein A-I in Cholesterol Efflux
Authors: Asztalos Bela , Zhang Wenwu , Roheim Paul S. , Wong Laurence
Published: June 2011

Abstract
This article characterizes products formed by the interaction of purified apolipoprotein (apo) A-I and human fibroblasts. Fibroblasts were incubated with different concentrations of purified apoA-I (1 to 30 μg/mL) in tissue culture medium for different periods of time (0 to 24 hours). The medium was then characterized by one- (agarose) and two-dimensional (agarose : polyacrylamide nondenaturing gradient gel) electrophoresis. At any given concentration of apoA-I, the rate of cellular cholesterol efflux appeared linear over 24 hours. Incubating purified apoA-I with fibroblasts for 4 hours, we detected five pre-α lipoproteins with particle sizes between 114 and 684 kDa. Formation of pre-α lipoproteins was concentration-dependent. At low concentrations (below 5 μg/mL apoA-I), all purified apoA-I (with pre-β mobility) was converted to pre-α lipoproteins. At higher concentrations (greater than 5 μg/mL apoA-I), more apoA-I remained with pre-β mobility. The pre-α lipoproteins were characterized by colocalization of apoA-I particles with
14
C-cholesterol and
32
P-phospholipids. Results showed that the pre-α particle of lowest molecular weight contained phospholipid and apoA-I but no cholesterol. The remaining pre-α particles contained all three substances. When pre-α particles were subjected to ultracentrifugation, all particles floated at d<1.21 g/mL with some of the smallest phospholipid apoA-I only particles being present in the d>1.21 g/mL fraction. Based on these results, we postulated that in the first stages of reverse cholesterol transport, pre-α lipoproteins are formed by the interaction of lipid free apoA-I and peripheral cells.
HDL Deficiency in Genetically Engineered Mice Requires Elevated LDL to Accelerate Atherogenesis
Authors: Hughes Steven D. , Verstuyft Judy , Rubin Edward M.
Published: June 2011

Abstract
In humans, a low HDL concentration is one of the strongest indicators of increased risk for coronary heart disease. Apolipoprotein A-I (apo A-I) synthetic defects result in extremely low HDL levels and are frequently although not invariably associated with premature atherosclerosis. To investigate atherosclerosis susceptibility associated with HDL deficiency alone and in combination with other risk factors, such as high levels of LDL, we have quantified diet-induced atherogenesis in a series of genetically engineered mice, including mice with low HDL levels due to targeted disruption of both apo A-I alleles (AI KO mice), mice with high LDL levels due to expression of a human apolipoprotein B transgene (Btg mice), and mice with combined high LDL and low HDL levels due to the presence of the human apo B transgene and apo A-I knockout alleles, respectively (AI KO/Btg mice). After exposure to an atherogenic diet, AI KO and control mice had negligible lesions. All mice expressing the apo B transgene developed extensive lesions, but AI KO/Btg mice developed significantly larger lesions than Btg mice: 56, 260±4630 μm
2
for AI KO/Btg (n=27) versus 38, 120±3350 μm
2
for Btg mice (n=19) (
P
<.02). Results of this study, consistent with several human epidemiological studies, indicate that HDL deficiency in the mouse does not by itself lead to the development of atherosclerosis but does increase atherosclerosis susceptibility when accompanied by other risk factors, in this case elevated LDL.
Inhibition of Inducible Nitric Oxide Synthase Restores Endothelium-Dependent Relaxations in Proinflammatory Mediator-Induced Blood Vessels
Authors: Kessler Paul , Bauersachs Johann , Busse Rudi , Schini-Kerth Valerié B.
Published: June 2011

AbstractEndothelium-dependent relaxations mediated by nitric oxide (NO) are attenuated in arteries exposed to proinflammatory mediators. Because proinflammatory mediators stimulate the expression of the inducible NO synthase (iNOS) in vascular cells, the role of iNOS-derived NO in the impaired endothelium-dependent relaxation was examined in arterial ring preparations. Exposure of rabbit carotid arteries to interleukin-1 β (IL-1 β; 100 U/mL for 7 hours) and porcine coronary arteries to a combination of tumor necrosis factor-α (1000 U/mL), interferon-γ (500 U/mL), and lipopolysaccharide (10 μg/mL) for 15 hours (conditions that are associated with iNOS expression) markedly attenuated relaxations to receptor-dependent agonists, whereas those to the calcium ionophore A23187 and sodium nitroprusside were virtually unchanged. The impaired relaxation was not associated with a reduced level of the constitutive endothelial NOS (cNOS) but was accompanied by a reduced formation of biologically active NO as assessed in a bioassay system. The attenuated relaxation of carotid arteries to acetylcholine was not affected by superoxide dismutase and was neither found in arteries exposed to IL-1 β for only 15 minutes nor in IL-1 β-treated arteries for 7 hours followed by a 17-hour incubation period without the cytokine. Furthermore, no impaired relaxation was found in rings exposed to IL-1 β in combination with either cycloheximide orN-α-tosyl-l-lysine chloromethyl ketone or pyrrolidine dithiocarbamate, treatments that prevent iNOS expression. In addition, selective inhibition of iNOS withS-methylisothiourea (10 μmol/L) completely restored acetylcholine-induced relaxations.

These findings indicate that the continuous generation of NO induced by proinflammatory mediators plays a major role in the inhibition of endothelium-dependent relaxation, most likely by impairing a step in the signal transduction cascade that links activation of endothelial receptors to the calcium-calmodulin-dependent activation of NOS.
Behaviorally Elicited Heart Rate Reactivity and Atherosclerosis in Ovariectomized Cynomolgus Monkeys (
Macaca fascicularis
)
Authors: Manuck Stephen B. , Adams Michael R. , McCaffery Jeanne M. , Kaplan Jay R.
Published: June 2011

Abstract
It has been hypothesized that atherogenesis is accelerated among individuals who exhibit heightened cardiovascular reactions to psychologic stress. We have reported previously that the coronary atherosclerosis of cholesterol-fed, male and reproductively intact (premenopausal) female cynomolgus monkeys was exacerbated in animals that experienced the largest heart rate (HR) reactions to a fear-eliciting laboratory stressor. In this article, we report a similar relationship among 20 female monkeys that were rendered estrogen-deficient (by ovariectomy) and subsequently treated with replacement of both estrogen and progesterone. At the beginning of a 30-month study period, animals were fitted with ECG telemetry devices, and their HRs were recorded under baseline and stressed conditions. Stress HR measurements were obtained during a standard challenge involving threatened capture and physical handling of the animals. As part of a related experiment, monkeys were then ovariectomized and, for the remainder of the study, administered 17β-estradiol (continuously) and progesterone (cyclically) by subcutaneous Silastic implant (Dow Corning). Animals consumed a cholesterol-containing diet throughout, and HR measurements were repeated in the 24th month. At necropsy, the magnitude of animals’ HR responses to stress correlated significantly with intimal area measurements in the left anterior descending and circumflex coronary arteries (
r
=.59 and
r
=.57, respectively;
P
<.009). This association was due to a marked exacerbation of coronary atherosclerosis in animals comprising the upper third of the reactivity distribution. Although total and HDL cholesterol concentrations also covaried with HR reactivity, the greater atherosclerosis of “high” HR reactors persisted after statistical adjustment for concomitant variability in plasma lipids. HR reactivity was unrelated to blood pressure, body weight, or social behavior.
Augmented Adenovirus-Mediated Gene Transfer to Atherosclerotic Vessels
Authors: Ooboshi Hiroaki , Rios C. David , Chu Yi , Christenson Stuart D. , Faraci Frank M. , Davidson Beverly L. , Heistad Donald D.
Published: June 2011

Abstract
Vascular endothelium is an important target for gene transfer in atherosclerosis. In this study, we examined gene transfer to normal and atherosclerotic blood vessels from two species, using an organ culture method. Using normal aorta, we determined optimal dose, duration of exposure to adenovirus, and duration of incubation of vessels in tissue culture. Aortas from normal and atherosclerotic monkeys were cut into rings and incubated for 2 hours with a recombinant adenovirus, carrying the reporter gene for β-galactosidase driven by a cytomegalovirus (CMV) promoter. After 20 hours of incubation, transgene expression was assessed with a morphometric method after histochemical staining and a chemiluminescent assay of enzyme activity. Expression of β-galactosidase after histochemical staining, expressed as percentage of total cells, was similar in adventitial cells of normal monkeys (21±4%, mean±SE%) and atherosclerotic monkeys (25±12%). Transgene expression in endothelium was higher in atherosclerotic than in normal vessel (53±3% versus 27±7%,
P
<.05). Chemiluminescent assay indicated greater β-galactosidase activity (2.5±0.6 mU/mg of protein) in the intima and media of atherosclerotic than normal vessels (0.6±0.2 mU/mg of protein,
P
<.05). Aortas from normal (n=6) and atherosclerotic (n=5) rabbits also were examined. Transgene expression (after histochemical staining) in endothelium was much greater in atherosclerotic than normal rabbits (39±3% versus 9±2%,
P
<.05) and expression in adventitial cells was similar (normal 23±2%, atherosclerotic 24±4%). Chemiluminescent assay indicated greater β-galactosidase activity (1.2±0.4 mU/mg of protein) in the intima and media of atherosclerotic than normal vessels (0.2±0.1 mU/mg protein,
P
<.05). These findings suggest that an adenoviral vector with a CMV promoter provides similar transgene expression in adventitia of both normal and atherosclerotic vessels. Gene transfer to the endothelium was much more effective in atherosclerotic than in normal vessels. Thus it may be possible to achieve greater transgene expression in atherosclerotic than in normal arteries.
The Antiadhesive and Antithrombotic Effects of the Nitric Oxide Donor SIN-1 Are Combined With a Decreased Vasoconstriction in a Porcine Model of Balloon Angioplasty
Authors: Provost Patrick , Tremblay Johanne , Merhi Yahye
Published: June 2011

Abstract
Nitric oxide has been reported to modulate platelet and neutrophil interactions with the arterial wall. In this study, we investigated the effects of the nitric oxide donor 3-morpholino-sydnonimine (SIN-1) on platelet and neutrophil adhesion, and the vasomotor response, in a porcine model of angioplasty. Carotid arterial injury was produced by balloon dilation in control (n=10) and treated (SIN-1; 10 μg/kg + 1 μg/kg/min, IV) (n=8) pigs. At the site of deep arterial injury, the average platelet adhesion was 53.6±11.3×10
6
/cm
2
in the control animals and was significantly inhibited by more than 70%, to 15.1±4.1×10
6
/cm
2
(
P
<.01), by SIN-1. Neutrophil adhesion was also decreased by SIN-1, from 255.9±29.7 to 101.8±19.7×10
3
/cm
2
(
P
<.001). Mural thrombosis was found in 12 (71%) of the 17 injured arteries in the control group but in only 2 (17%) of the 12 injured arteries in the SIN-1–treated group (
P
<.05). Concomitantly, SIN-1 reduced platelet and neutrophil adhesion to the site of endothelial injury distally. The internal diameter of the carotid arteries was similar between the two groups before dilation but was 40% greater at the site of endothelial injury distally in SIN-1–treated animals after dilation (
P
<.05), as compared with controls. Accordingly, postangioplasty vasoconstriction was significantly attenuated from 46.3±2.9% in control pigs to 32.5±4.8% (
P
<.05) in SIN-1–treated animals. The beneficial effects of SIN-1 were associated with inhibition of neutrophil-mediated whole blood aggregation and of neutrophil-endothelium interactions. The potent antiadhesive and antithrombotic properties of SIN-1 in vivo were confirmed in ex vivo superfusion experiments. These results indicate that administration of a nitric oxide donor may be effective in preventing the acute pathophysiological responses to arterial injury by angioplasty.
Thermal Preconditioning Before Rat Arterial Balloon Injury
Authors: Neschis David G. , Safford Shawn D. , Raghunath Puthiyaveettil N. , Langer David J. , David Mary L. , Hanna Abigail K. , Tomaszewski John E. , Kariko Katalin , Barnathan Elliot S. , Golden Michael A.
Published: June 2011

Abstract—Heat shock proteins (HSPs) are a family of highly conserved proteins, essential to cell survival, that are induced during times of physiological stress. These proteins, when induced, can provide tolerance to subsequent injury. Several studies have documented that HSPs play an important role in the response of vascular cells to injury or stress. Whether the vasculature itself can be effectively preconditioned before arterial injury is unknown. Vascular HSP induction by whole-body hyperthermia (WBH) was evaluated with regard to its effects on the vascular response to balloon injury. WBH treatment of Sprague-Dawley rats (colonic temperatures of 41 to 42°C for 15 minutes) resulted in maximal arterial HSP expression within 8 to 12 hours. Rats (male, 300 g, n=59) were randomly assigned to undergo either WBH or no treatment 8 hours before standard carotid balloon injury. At 14 (n=26) and 90 (n=21) days after balloon injury, histomorphometric analysis revealed a significant limitation of intimal accumulation in preconditioned arteries as compared to controls (intimal/medial area ratios±SEM: 14 days, 0.57±0.07 versus 0.86±0.08,P=0.01; 90 days, 0.78±0.12 versus 1.19±0.14,P<0.05). The medial cell proliferation index at 4 days (n=12) was significantly reduced in the treated group as well (3.6±0.9% versus 7.2±1.3%,P<0.05). Conversely, the mean total cell number in the media of heated arteries was higher (393±20 versus 328±17,P<0.05). Vascular preconditioning with brief WBH induces a heat shock response in the arterial wall that is associated with a significant and sustained reduction in intimal accumulation. This effect appears to be due in part to preservation of medial cell integrity and limitation of the proliferative response. These results suggest that thermal preconditioning of vascular tissue may be an effective strategy to improve long-term results after revascularization procedures.
Absence of Age-Related Increase in Central Arterial Stiffness in Physically Active Women
Authors: Tanaka Hirofumi , DeSouza Christopher A. , Seals Douglas R.
Published: June 2011

Abstract
—Increased arterial stiffness is thought to contribute to the increased incidence of cardiovascular disease with age. Little, however, is known about the influence of aging on central and peripheral arterial stiffness in females. Moreover, it is unknown whether physical activity status influences age-related increases in arterial stiffness in females. Arterial pulse wave velocity (PWV) and augmentation index (AI, applanation tonometry) were measured in 53 healthy females, including 10 premenopausal (Pre-S) and 18 postmenopausal (Post-S) sedentary women, and 9 premenopausal (Pre-PA) and 16 postmenopausal (Post-PA) physically active women. In the sedentary women, there were no age-related differences in arterial blood pressure, but aortic PWV and carotid AI (measures of central arterial stiffness) were higher (
P
<.01) in Post-S versus Pre-S (1065±110 versus 690±80 cm/sec and 16.5%±1.8% versus 0.3%±1.6%, respectively); however, there were no significant differences in leg and arm PWV (measures of peripheral arterial stiffness). Systolic and mean arterial blood pressures were higher (
P
<.05) in Post-PA versus Pre-PA. Despite this and in contrast to the sedentary women, aortic PWV and AI were not different in Post-PA versus Pre-PA. Stepwise multiple regression indicated that maximal oxygen consumption, plasma total cholesterol, and plasma LDL-cholesterol were significant independent predictors and together explained up to 50% of the variability in central arterial stiffness. We concluded that (1) central, but not peripheral, arterial stiffness increases with age in sedentary healthy females in the absence of age-related increases in arterial blood pressure; (2) significant age-related increases in central arterial stiffness are not observed in highly physically active women; and (3) aerobic fitness and plasma total cholesterol and LDL-cholesterol levels are significant independent physiological correlates of central arterial stiffness in this population.
Endogenous Nitric Oxide Protects Against Thromboembolism in Venules But Not in Arterioles
Authors: Broeders Martijn A. W. , Tangelder Geert-Jan , Slaaf Dick W. , Reneman Robert S. , oude Egbrink Mirjam G. A.
Published: June 2011

Abstract
—Because nitric oxide (NO) inhibits aggregation and adhesion of blood platelets, NO may play a role in platelet–vessel wall interactions. Therefore, the purpose of this study was to investigate the involvement of endogenous NO in thromboembolic processes, as induced by wall puncture, in rabbit mesenteric arterioles and venules (diameters 20 to 43 μm). In venules, inhibition of NO synthase by superfusion of the mesentery with
N
ω
-nitro-
l
-arginine (L-NA; 0.1 mmol/L) significantly increased the duration of embolization (from 50 seconds to 511 seconds) and the number of emboli produced (from 2 to 11 emboli per vessel), while the median period of time needed to produce an embolus was not influenced. On the contrary, in arterioles, L-NA had no significant effect on embolization (duration of embolization: 426 seconds in the control and 382 seconds in the L-NA group, with 20 and 12 emboli per vessel, respectively). Addition to the L-NA superfusate of L-arginine (L-ARG; 1 mmol/L), the active precursor for endogenous NO synthesis, resulted in a complete reversal of the L-NA effects in venules, while addition of the inactive D-arginine (D-ARG; 1 mmol/L) had no effect. Addition of L-ARG and D-ARG had no significant effect in arterioles. Addition to the L-NA superfusate of the exogenous NO donor sodium nitroprusside (0.1 μmol/L) also resulted in reversal of the L-NA effects in venules, while in arterioles, it slightly but significantly decreased embolization duration. The differences in effect of L-NA on embolization between arterioles and venules were not caused by differences in fluid dynamic conditions. It is concluded that the role of endogenous NO in inhibiting thromboembolic processes is more important in venules than in arterioles.