bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Expression of a P
2T
ADP Receptor in
Xenopus
Oocytes Injected With Megakaryocyte (CMK 11-5) RNA
Authors: Greco Nicholas J.
Published: June 2011

Abstract
Since the P
2T
purinergic (ADP) receptor is unique to the megakaryocytic/platelet lineage, cells of this lineage were screened for the relative effects of ADP and ATP in intracellular Ca
2+
levels. Like platelets, CMK 11-5 cells responded with an increase in intracellular Ca
2+
mobilization in response to ADP but not to ATP or adenosine. In contrast, both nucleotides increased intracellular Ca
2+
mobilization in the megakaryoblastic cell lines MO7E and Meg-01, indicating that they contain P
2Y
receptors or a mixed complement of purinergic receptors. Pharmacological responsiveness of CMK 11-5 cells to nucleotides paralleled those of platelets, in which ADP and ADP-α-S are active as agonists and ATP and ATP-α-S are inactive as agonists but act as antagonists. [
3
H]ADP and
35
S-ATP-α-S bound to CMK 11-5 cells at a high-affinity site (
K
d1
and
K
i1
, 262 and 125 nmol/L, respectively) and a low-affinity site (
K
d2
and
K
i2
, 10 100 and 5400 nmol/L, respectively) with 2×10
6
to 6×10
6
sites per cell. ADP bound at both sites was competed with ADP, ATP, and ATP-α-S with affinities in a rank order similar to that found for platelets (ATP-α-S≈ATP≈ADP≥ADP-β-S≈adenosine), suggesting the presence of a P
2T
receptor on CMK 11-5 cells. Photoaffinity labeling of intact CMK 11-5 cells with
35
S-ATP-α-S resulted in the labeling of the α-subunit of GP IIb as found with platelets, although this was confirmed to be independent of ADP receptors. After RNA from CMK 11-5 cells was microinjected into
Xenopus
oocytes, only ADP and ADP-α-S stimulated
45
Ca
2+
efflux, which was not observed with ATP, 2-methylthio-ATP, α,β-methylene-ATP, ATP-γ-S, ATP-α-S, or adenosine. In addition, incubation of RNA-injected oocytes with ATP or ATP-α-S but not adenosine blocked the
45
Ca
2+
response to ADP. These experiments demonstrate that a nascent receptor that responded specifically to ADP but not to other P
1
, P
2Y
, P
2X
, and P
2U
agonists was expressed in functional form on
Xenopus
oocytes.
Synthesis and Secretion of von Willebrand Factor and Fibronectin in Megakaryocytes at Different Phases of Maturation
Authors: Schick Paul K. , Walker Jean , Profeta Bernadette , Denisova Lyudmila , Bennett Vickie
Published: June 2011

Abstract
Our goals have been to define the biochemical characteristics of megakaryocytes during maturation that are critical for platelet assembly and release into the circulation and to introduce biochemical markers for megakaryocytes. To achieve these goals, we have studied fibronectin (FN) and von Willebrand factor (vWF), which are large adhesive proteins that are synthesized by megakaryocytes, stored in alpha granules, and thought to have a fundamental role in hemostasis. The study demonstrated that vWF is primarily synthesized in mature megakaryocytes, which synthesized 7.5 times more vWF than immature megakaryocytes. Brefeldin A, which blocks the exit of proteins from the rough endoplasmic reticulum (RER), inhibited the formation of vWF multimers but did not affect the synthesis of monomers and dimers in mature megakaryocytes. These data are consistent with the formation of vWF dimers in the RER and the assembly of vWF multimers in the trans- and post-golgi. The synthesis of both the 260-kD and 275-kD pro-vWF was detected. However, the synthesis of 275-kD pro-vWF and 220-kD mature vWF was only evident after 2 hours, suggesting that the transit time of nascent vWF through the RER is about 2 hours. Constitutive secretion of vWF was demonstrated in megakaryocytes. About 14.5% and 4.6% of synthesized vWF was secreted by mature and immature megakaryocytes, respectively. In contrast, the synthesis of FN monomers and dimers was established in immature megakaryocytes, and their synthesis in mature megakaryocytes was very similar. Constitutive secretion of FN was not seen in megakaryocytes. Brefeldin A did not inhibit the synthesis of FN dimers; thus, formation of FN dimers occurs in the RER. The demonstration that vWF and FN are synthesized at different phases of megakaryocyte maturation and that only vWF is constitutively secreted by megakaryocytes provides new information relevant to alpha granule formation and possibly bone marrow matrix assembly.
Associations Between Diet and the Hyperapobetalipoproteinemia Phenotype Expression in Children and Young Adults
Authors: Nuotio Ilpo O. , Raitakari Olli T. , Porkka Kimmo V.K. , Räsänen Leena , Moilanen Teemu , Viikari Jorma S.A.
Published: June 2011

Abstract
The effect of diet on blood lipids has been under intensive study during recent decades. However, diet in the context of the hyperapobetalipoproteinemia (hyperapoB) phenotype has received less attention. The hyperapoB phenotype is commonly encountered in patients with premature coronary heart disease. It is defined as a combination of an increased concentration of apolipoprotein B (apo B), a normal concentration of LDL cholesterol (LDL-C), and as a result, a low LDL-C/apo B ratio. We studied the associations between diet and blood lipids in a cohort of 534 children and young adults 9 to 24 years old. The ratio of polyunsaturated to saturated fats (P/S ratio) correlated (
r
=−0.19,
P
<.001) with the LDL-C/apo B ratio. This association was also found when the model was adjusted with triglycerides (
r
=−0.24,
P
<.001). A change in the P/S ratio from 0.10 to 0.60 corresponded to a decrease of 0.12 in the LDL-C/apo B ratio, and in the highest apo B decile, the P/S value was higher in hyperapoB individuals (0.33) than in others (0.28,
P
=.019). Our results imply that the fatty acid composition of the diet may be one of the environmental factors that influence the hyperapoB phenotype expression.
Shared and Unique Genetic Effects Among Seven HDL Phenotypes
Authors: Comuzzie Anthony G. , Rainwater David L. , Blangero John , Mahaney Michael C. , VandeBerg John L. , MacCluer Jean W.
Published: June 2011

Abstract
The purpose of this study was to investigate the genetic control of various HDL measures and to determine the proportion of genetic variance explained by shared genes (ie, pleiotropy) and the proportion unique to each trait. The data used were drawn from large, randomly ascertained pedigrees of Mexican Americans participating in the San Antonio Family Heart Study. Data were available for 655 individuals (258 men and 397 women) in 26 families. We performed a multivariate quantitative genetic analysis to simultaneously estimate both the additive genetic and random environmental correlations among seven HDL phenotypes. These seven HDL phenotypes can be divided into two categories: measures of concentration and estimates of particle size. Concentration was measured for apo A-I, apo A-II, esterified cholesterol, and unesterified cholesterol, and particle size was estimated for apo A-I, apo A-II, and esterified cholesterol. The heritabilities (h
2
) for each of the seven traits were significantly greater than zero (
P
<.05) and ranged from 0.2 to 0.6. When considered in a pairwise fashion, all combinations of these traits showed marked genetic correlations (ρ
G
=0.33 to 0.87) and all were significantly greater than zero (
P
<.05), indicative of pleiotropic effects. However, we found substantial unique genetic variance for each of these traits even after accounting for the effects shared in common with all the remaining measures. We conclude that the genetic variation in these HDL phenotypes is a result of the action of common as well as unique genes.
Subjects With ApoA-I(Lys
107
→0) Exhibit Enhanced Fractional Catabolic Rate of ApoA-I in Lp(AI) and ApoA-II in Lp(AI With AII)
Authors: Tilly-Kiesi Marju , Lichtenstein Alice H. , Ordovas Jose M. , Dolnikowski Gregory , Malmström Raija , Taskinen Marja-Riitta , Schaefer Ernst J.
Published: June 2011

Abstract
Our purpose was to examine HDL metabolism in a Finnish kindred with a 3-bp deletion in the apolipoprotein (apo) A-I gene, resulting in a deletion of Lys
107
in the mature apoA-I. Patients with this mutation [apoA-I(Lys
107
→0)] have reduced plasma HDL cholesterol and lipoprotein (AI with AII) [Lp(AI w AII)] concentrations, but not Lp(AI) levels, compared with unaffected family members. Using primed constant infusions of [5,5,5-
2
H
3
]leucine, we determined the residence time (RT) and absolute production rate (APR) of apoA-I and apoA-II entering plasma in two subpopulations of HDL particles: [Lp(AI) and Lp(AI w AII)] in three patients heterozygous for apoA-I(Lys
107
→0) and in seven healthy control subjects. In patients, the mean RT of apoA-I in Lp(AI) (3.75±1.68 days) was less than half that observed in control subjects (8.01±2.51 days,
P
<.05). The mean RT of apoA-I in Lp(AI w AII) was also lower in patients than in control subjects, but differences were not statistically significant (4.72±2.42 versus 6.50±2.19 days). The mean RT of apoA-II in Lp(AI w AII) was significantly lower in patients (5.24±1.65 days) than in control subjects (9.64±3.57 days,
P
<.05). The APR of apoA-I into Lp(AI) was twofold higher in patients (5.9±2.1 mg·kg
−1
·d
−1
) than in control subjects (2.5±0.9,
P
<.05). The APRs of apoA-I and apoA-II into Lp(AI w AII) were similar in patients and control subjects. Our results are consistent with the concept that patients heterozygous for the apoA-I(Lys
107
→0) mutation have enhanced fractional catabolism of apoA-I and apoA-II in both HDL subspecies, especially in Lp(AI), and an increase in apoA-I production only into Lp(AI), which may be compensatory. Therefore, only their Lp(AI w AII) levels are decreased.
Effective Lowering of Plasma, LDL, and Esterified Cholesterol in LDL Receptor–Knockout Mice by Adenovirus-Mediated Gene Delivery of ApoB mRNA Editing Enzyme (Apobec1)
Authors: Teng BaBie , Ishida Brian , Forte Trudy M. , Blumenthal Scott , Song Li-Zhen , Gotto, Jr Antonio M. , Chan Lawrence
Published: June 2011

Abstract
Adenovirus-mediated gene delivery of apolipoprotein (apo)B mRNA editing enzyme (AvApobec1) was used to study the effect of apoB mRNA editing on apoB production in homozygous LDL receptor–deficient (LDLR-/-) mice. Intravenous injection of AvApobec1 into these mice resulted in a >80% decrease in plasma apoB-100 with a concomitant increase in plasma apoB-48 level. The plasma apoE level also increased. In all cases, total plasma apoB (apoB-100 + apoB-48) decreased by 60% at day 5 and remained ≈40% lower in AvApobec1-treated compared with control vector Av1LacZ4–treated animals at day 12. On day 12, total plasma cholesterol decreased by 29% in male mice and 18% in female mice that were transduced with AvApobec1. This was reflected in a reduction in apoB-containing lipoprotein cholesterol, which decreased by 34% and 27% in male and female mice, respectively. Apobec1 gene transfer also decreased the cholesteryl ester contents in the LDL fraction, which were 16%, 22%, and 22% in female and 20%, 20%, and 15% in male animals on days 5, 7, and 12, respectively, compared with Av1LacZ controls with 29%, 32%, and 33%, respectively, in female and 29%, 38%, and 36%, respectively, in male animals. Nondenaturing gradient gel electrophoresis indicated almost complete elimination of LDL particles of 29, 27, and 25 nm at days 7 and 12. We conclude that in the absence of a functioning LDL receptor, hepatic overexpression of Apobec1 is highly efficient in lowering plasma apoB-100 levels, leading to the almost complete elimination of LDL particles and a reduction in LDL cholesterol and cholesteryl ester content.
Acidic and Basic Fibroblast Growth Factors Suppress Transcriptional Activation of Tissue Factor and Other Inflammatory Genes in Endothelial Cells
Authors: Pendurthi Usha R. , Williams J. Todd , Rao L. Vijaya Mohan
Published: June 2011

Abstract
Tissue factor (TF) is a transmembrane receptor that serves as a cofactor for factor VIIa and initiates the extrinsic pathway of blood coagulation. Under normal physiological conditions, TF is expressed in extravascular and perivascular cells but not in vascular endothelial cells and monocytes. TF can be induced in these cells by inflammatory regulators and other stimulators, such as LPS, thrombin, oxidized lipoproteins, and certain growth factors. An earlier study showed that growing primary cultures of human umbilical vein endothelial cells (HUVECs) with endothelial cell growth supplement (ECGS) and heparin had impaired the ability of monolayers to express surface membrane TF activity after perturbation. The mechanism by which ECGS suppressed TF activity was not known. In the present study, we investigated the effect of recombinant acidic and basic fibroblast growth factors (aFGF and bFGF) on the induction of TF in a HUVEC cell line and a fibroblast cell line. Both aFGF and bFGF suppressed the phorbol myristate acetate–induced expression of TF in endothelial cells but not the serum-induced expression of TF in fibroblast cells. Diminished expression of the cell surface TF activity observed in endothelial cells grown with aFGF or bFGF was due to the accumulation of a lower number of TF mRNA transcripts. TF mRNA stability was not altered in HUVECs grown with aFGF or bFGF. Nuclear run-on experiments revealed that the transcription of TF and several other genes that play an important role in inflammation and angiogenesis was reduced in the endothelial cells that were cultured with aFGF or bFGF. The diminished expression of TF may be part of a generalized response of endothelial cells to FGF that facilitates migration of endothelial cells during angiogenesis.
Impact of a Combination of a Calcium Antagonist and a β-Blocker on Cell- and Copper-Mediated Oxidation of LDL and on the Accumulation and Efflux of Cholesterol in Human Macrophages and Murine J774 Cells
Authors: Lesnik Philippe , Dachet Christiane , Petit Laure , Moreau Martine , Griglio Sabine , Brudi Philippe , Chapman M. John
Published: June 2011

Abstract
Calcium antagonists and β-blockers may retard or inhibit atherogenesis. In the absence of data pertaining to the potential cardioprotective action of an association of such agents, we have investigated the impact of nifedipine and atenolol, alone or in combination, on the capacity of monocyte-macrophages (ex vivo) and copper ions (in vitro) to oxidize LDL and on intracellular metabolism and efflux of free and esterified forms of cholesterol in human macrophages and foam cells. At concentrations up to 100 μmol/L, atenolol had no effect on the oxidative resistance of LDL; on the contrary, nifedipine displayed a significant dose-dependent capacity to protect LDL during copper-mediated oxidation (100 μmol/L;
P
<.001). Using a DPPH radical generating system, nifedipine was shown to exert free radical–trapping activity (molar ratio of scavenging activity, nifedipine:α-tocopherol, 1:114). The addition of atenolol to nifedipine was without effect on the antioxidant activity of the calcium antagonist. In experiments in which oxidative modification was mediated by monocyte-macrophages, nifedipine but not atenolol conserved its antioxidant capacity. Furthermore, we demonstrated that association of atenolol with nifedipine did not modify the antioxidant properties of nifedipine itself. Using a human monocyte-derived macrophage culture system, nifedipine, atenolol, or a combination of the two drugs was ineffective in inhibiting foam cell formation induced by acetylated LDL or oxidized LDL. However, atenolol (100 μmol/L) increased cellular accumulation of cholesteryl ester (+17%;
P
<.05), whereas nifedipine (100 μmol/L) decreased total cholesterol (−37.4%;
P
<.05) accumulation induced by acetylated LDL in the mouse macrophage cell line J774. A combination of the two drugs neutralized these antagonistic effects. None of these results were reproduced during the oxidized LDL–induced transformation of murine J774 cells into foam cells. Furthermore, cholesterol efflux from preloaded human macrophages was equally unaffected by the addition of the drugs alone or in combination. It therefore seems unlikely that the beneficial effect of atenolol on coronary heart disease is mediated by changes in either LDL oxidizability or cholesterol metabolism in human macrophages and foam cells. Our findings with nifedipine suggest, however, that this calcium antagonist may potentially exert antiatherosclerotic properties via a reduction of the oxidative modification of LDL, thereby affecting a reduction in foam cell formation and in the pathophysiological cellular activities of oxidized lipids, rather than by inducing a direct reduction in cholesterol accumulation in human foam cells of macrophage origin.
Alcohol Increases Plasma Levels of Cholesterol Diet–Induced Atherogenic Lipoproteins and Aortic Atherosclerosis in Rabbits
Authors: Shaish Aviv , Pape Michael , Rea Thomas , Srivastava Rai Ajit K. , Latour Mickey A. , Hopkins Dan , Schonfeld Gustav
Published: June 2011

AbstractThe purpose of the present study was to reexamine the relationship between alcohol and atherosclerosis. Two experiments were performed: The first contained three groups of New Zealand White (NZW) female rabbits. The control group was fed a cholesterol-containing liquid diet and the other two groups were fed the same diet with either 20% or 30% of the calories supplied by alcohol. The second experiment had two treatments: one control group and another group fed a 10% alcohol diet. In experiment 1, alcohol at the 20% and 30% levels increased VLDL and LDL but not HDL compared with levels in control rabbits. Hepatic mRNA levels of apolipoprotein (apo) A-I, apoB, and 7α-hydroxylase were not affected by alcohol. However, the LDL-receptor mRNA was decreased to half of control values by either 20% or 30% alcohol. Lesion areas and aortic cholesterols were significantly increased in the 20% and 30% alcohol–treated groups. Also, significant correlations were found between plasma cholesterol levels and total lesion area or lesion cholesterol contents. In experiment 2, the 10% alcohol–treated rabbits showed no differences in circulating lipoproteins, LDL-receptor mRNA, or lesion formation above that observed in controls. These experiments suggest that alcohol substituted at 20% or 30% of the dietary calories induces hypercholesterolemia and more aortic atherosclerotic lesions. The alcohol-induced accumulation of VLDL and LDL was accompanied by low hepatic LDL-receptor mRNA levels, suggesting that alcohol may affect LDL-receptor expression and rates of lipoprotein clearance, but more experiments are needed to evaluate this possibility.
Fibrinolytic Activity Is Similar in Physically Active Men With and Without a History of Myocardial Infarction
Authors: Fernhall Bo , Szymanski Linda M. , Gorman Patrick A. , Milani James , Paup Donald C. , Kessler Craig M.
Published: June 2011

Abstract
The purpose of this study was to evaluate fibrinolytic potential at rest and after a fibrinolytic stressor in men with a history of myocardial infarction (MI) compared with an age- and activity-matched group of men without coronary artery disease (CAD). All men were currently enrolled in exercise programs. Tissue-type plasminogen activator (TPA) and plasminogen activator inhibitor 1 (PAI-1) activity and antigen levels were measured at rest and after a maximal exercise test. A 2×2 (group×time) ANOVA with repeated measures was used to evaluate fibrinolytic potential. Bivariate regressions were conducted to evaluate relations between fibrinolytic potential and maximal oxygen uptake (V̇O
2max
). Age was similar between groups (CAD, 57.5±6.6; non-CAD, 58.1±7.3 years); however, V̇O
2max
was higher in non-CAD subjects (36.2±6.2 vs 27.5±5.9 mL·kg
−1
·min
−1
). Mean±SEM resting TPA and PAI-1 activities were similar between CAD and non-CAD subjects (TPA, 2.8±0.2 vs 2.8±0.2 IU/mL; PAI-1, 15.9±3.1 vs 13.1±4.1 AU/mL). Both groups showed similar significant increases in TPA activity with exercise (
P
<.05), and postexercise TPA activity was also similar (CAD, 9.1±2.0 IU/mL; non-CAD, 11.7±2.6 IU/mL). Both groups also showed similar significant decreases in PAI-1 activity with exercise (
P
<.05) and no differences in postexercise PAI-1 activity (CAD, 13.2±2.5 AU/mL; non-CAD, 10.4±3.6 AU/mL). Significantly higher resting TPA antigen levels were seen in CAD (14.8 ng/mL) than non-CAD (10.2 ng/mL) subjects (
P
<.05), but neither group showed significant changes with exercise (CAD, 12.9 ng/mL; non-CAD, 11.8 ng/mL). Resting PAI-1 antigen was similar in the two groups (CAD, 71.4 ng/mL; non-CAD, 74.2 ng/mL) and did not significantly change with exercise (CAD, 77.9 ng/mL; non-CAD, 72.3 ng/mL). V̇O
2max
was positively correlated with postexercise TPA activity (
r
=.52,
P
<.05) and negatively correlated with resting TPA antigen (
r
=−.43,
P
<.05). Resting TPA antigen was also directly correlated with body mass index (
r
=.63,
P
<.05). The finding that functional fibrinolytic activity was not different in physically active men with and without CAD contrasts with previous reports. This suggests that matching subjects on the bases of age and habitual physical activity status and controlling exercise intensity are important factors to consider when evaluating fibrinolytic potential.
Association of Serum Vitamin Levels, LDL Susceptibility to Oxidation, and Autoantibodies Against MDA-LDL With Carotid Atherosclerosis
Authors: Iribarren Carlos , Folsom Aaron R. , Jacobs, Jr David R. , Gross Myron D. , Belcher John D. , Eckfeldt John H.
Published: June 2011

Abstract
Oxidative modification of LDL is believed to be a crucial step in atherosclerosis. Thus, antioxidant vitamins may have a role in the prevention of coronary disease. We examined the cross-sectional association of serum vitamin levels, the susceptibility of LDL to hemin-induced oxidation (lag phase to conjugated diene formation), and the malondialdehyde-LDL (MDA-LDL) to native LDL radioactivity binding ratio with carotid intima-media thickness (IMT), a measure of asymptomatic early atherosclerosis. The participants in this observational study were 231 asymptomatic age-, sex-, race-, and field center–matched case-control pairs selected from the Atherosclerosis Risk in Communities (ARIC) study cohort on the basis of B-mode carotid artery ultrasonograms obtained from 1986 through 1989. Cases exceeded the 90th percentile of IMT, and control subjects were below the 75th percentile of IMT for all arterial segments. Biochemical analyses were performed on fasting frozen (−70°C) serum specimens collected from 1990 through 1992. In conditional logistic regression adjusting for age, blood storage time, total cholesterol, and log-triglyceride concentrations, serum β-cryptoxanthin and lutein plus zeaxanthin levels were inversely related to the extent of atherosclerosis (odds ratio [OR] per 1-SD increase: 0.75, 95% confidence interval [CI]: 0.59-0.94; and OR per 1-SD increase: 0.76, 95% CI: 0.59-0.95, respectively). Increases in α-carotene and lycopene were associated with nonsignificantly lower odds of being a case, whereas β-carotene, retinol, and α-tocopherol were unrelated to IMT. Although not reaching statistical significance, the lag phase and autoantibodies against MDA-LDL were positively associated with asymptomatic atherosclerosis. After adjustment for potential confounders, only the inverse association of lutein plus zeaxanthin with asymptomatic atherosclerosis was maintained. This study supports a modest inverse association between circulating levels of some carotenoids, particularly lutein plus zeaxanthin, and carotid IMT. These findings suggest that these carotenoid compounds (regarded as biomarkers of fruit and vegetable intake) may be important in early stages of atherosclerosis.
Increased Immunolocalization of Paraoxonase, Clusterin, and Apolipoprotein A-I in the Human Artery Wall With the Progression of Atherosclerosis
Authors: Mackness Bharti , Hunt Roger , Durrington Paul N. , Mackness Michael I.
Published: June 2011

AbstractUsing immunolocalization techniques, we have shown that paraoxonase (Pon), clusterin, and apolipoprotein (apo) A-I accumulate in the artery wall during the development of atherosclerosis. In normal aortas (n=6) there were low levels of extracellular Pon, clusterin, and apoA-I immunoreactivity. The cytoplasm of smooth muscle cells in the media showed granular positivity for both Pon and apoA-I, indicating that these proteins were undergoing lysosomal degradation. This activity was also indicated by the presence of both intact and degradation products of Pon in smooth muscle cells as shown by Western blotting. With the progression of disease from fatty streaks (n=3) to advanced atherosclerosis (n=8) there was an increase in Pon, apoA-I, and clusterin immunoreactivity, indicating the increasing presence of these proteins with disease progression. These proteins are the components of a specific HDL subspecies that has been implicated in the prevention of peroxidative damage to phospholipids in LDL and membranes. The increase in Pon, clusterin, and apoA-I during the development of atherosclerosis may therefore represent a protective response to the oxidative stress associated with the development of atherosclerosis.
Bimodal Effects of Angiotensin II on Migration of Human and Rat Smooth Muscle Cells
Authors: Liu Guizben , Espinosa Emma , Oemar Barry S. , Lüscher Thomas F.
Published: June 2011

Abstract
Angiotensin II may be an important mediator of neointima formation in vascular disease. This study was designed to examine the mechanisms involved in angiotensin II-stimulated migration of human and rat aortic vascular smooth muscle cells (VSMCs). VSMCs were seeded in one corner of Nunc four-well culture chambers; angiotensin II within filter paper was glued onto the wall of the opposite side. After 48 hours of incubation in serum-free medium containing growth-arresting factor, migrated cells were counted using a light microscope. Angiotensin II (2×10
−11
to 2×10
−8
mol/L) increased migration of VSMCs in a concentration-dependent manner. Interestingly, at higher concentrations of angiotensin II (up to 2×10
−6
mol/L), migration was reduced to levels comparable with control levels. Losartan, an AT
1
receptor antagonist, prevented migration, while PD123319, an AT
2
receptor antagonist, had no significant inhibitory effect. Transforming growth factor-β1 (TGF-β1; 0.01 to 10.0 pg/mL) inhibited migration induced by angiotensin II (2×10
−8
mol/L) in a concentration-dependent manner. A neutralizing TGF-β antibody unmasked migratory effects of high concentrations of angiotensin II. Furthermore, angiotensin II (10
−6
mol/L) upregulated TGF-β1 mRNA levels fivefold in rat and fourfold in human VSMCs; this effect was prevented by losartan but not by PD123319. Thus, the effects of angiotensin II on migration of VSMCs are bimodal, ie, both migratory and antimigratory pathways are activated. Autocrine release of TGF-β1 induced by angiotensin II exerts an antimigratory effect in rat and human VSMCs. The AT
1
receptor is involved in regulation of both pathways.
Sex Differences in Visceral Fat Lipolysis and Metabolic Complications of Obesity
Authors: Lönnqvist Fredrik , Thörne Anders , Large Valerie , Arner Peter
Published: June 2011

Abstract
Cardiovascular complications of obesity are more common in men than women. Sex differences in visceral fat lipolysis may be of importance in this respect, since increased release of free fatty acids (FFAs) from visceral fat to the liver by the portal venous system has been thought to cause several metabolic complications due to obesity, such as hypertension, hyperlipidemia, and glucose intolerance. The aim of this study was to investigate sex differences in clinical characteristics and visceral fat mobilization in obesity. Obese subjects (22 male and 23 female) undergoing elective surgery were matched for body mass index and age. The males had both higher waist-to-hip ratio (WHR), sagittal diameter, blood pressure, fat-cell volume, plasma insulin, glucose, and triglyceride and lower HDL cholesterol levels than the females. The rate of norepinephrine-induced FFA and glycerol release was twofold higher in men (
P
=.02). No significant reutilization of FFA was observed. The difference in maximum norepinephrine-induced rate of lipolysis between men and women was independent of both WHR and sagittal diameter and was an independent regressor for levels of plasma glucose and plasma HDL cholesterol. Fat-cell volume was an independent regressor for plasma triglycerides and blood pressure. No sex differences in the lipolytic sensitivity to β
1
- or β
2
-adrenoceptor–specific agonists or in the antilipolytic effect of insulin were observed. However, the lipolytic β
3
-adrenoceptor sensitivity was 12 times higher (
P
=.004) and the antilipolytic α
2
-adrenoceptor sensitivity 17 times lower (
P
=.003) in men. Furthermore, lipolysis induced by agents acting at the adenylate cyclase and protein kinase A levels were almost twofold enhanced in men. However, no sex difference in maximum hormone-sensitive lipase activity was observed. In conclusion, in obesity, catecholamine-induced rate of FFA mobilization from visceral fat to the portal venous system is higher in men than women. This phenomenon is partly due to a larger fat-cell volume but also to a decrease in the function of α
2
-adrenoceptors, an increase in the function of β
3
-adrenoceptors, and an increased ability of cyclic AMP to activate hormone-sensitive lipase. These factors may contribute to gender-specific differences in metabolic and cardiovascular disturbances accompanied by obesity.
Connective Tissue Growth Factor
Authors: Oemar Barry S. , Lüscher Thomas F.
Published: June 2011

Abstract
Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted peptide, which is implicated in human atherosclerosis and fibrotic disorders such as systemic scleroderma. CTGF is a member of the peptide family that includes serum-induced immediate early gene products, a v-
src
–induced peptide, and a putative proto-oncogene. The CTGF gene family is a modular protein and is conserved throughout evolution. CTGF mRNA has been found in the human, mouse, chicken, frog, and fly. The functions of the CTGF gene family include embryogenesis, wound healing, and regulation of extracellular matrix production. Human CTGF is undetectable in normal blood vessels but overexpressed in atherosclerotic lesions, suggesting an important role in atherogenesis.
Hopelessness and 4-Year Progression of Carotid Atherosclerosis
Authors: Everson Susan A. , Kaplan George A. , Goldberg Debbie E. , Salonen Riitta , Salonen Jukka T.
Published: June 2011

Abstract
The importance of hope has long been recognized, whereas a lack of hope, or “giving up,” is generally believed to have a negative impact on psychological well-being and physical health. Recently, hopelessness has been identified as a strong, independent predictor of cardiovascular disease morbidity and mortality in both American and Finnish populations. In this study we examined the association between high levels of hopelessness and progression of carotid atherosclerosis in participants (n=942) in the Kuopio Ischemic Heart Disease Study, a population-based study of middle-aged men from eastern Finland who underwent carotid ultrasonography at baseline and 4 years later. Men reporting high levels of hopelessness at baseline had faster progression of carotid atherosclerosis, assessed by four measures of intima-media thickening (IMT), than men reporting low to moderate levels of hopelessness. Further analyses revealed significant interactions between hopelessness and initial level of atherosclerosis, such that the effects of high hopelessness on progression were greatest among men who had baseline mean IMT values at or above the median. Moreover, progression was greatest among men reporting high levels of hopelessness at both baseline and follow-up. Traditional coronary risk factors and use of cholesterol-lowering and antihypertensive medications did not account for much variance in the observed relationships. These findings indicate that hopelessness contributes to accelerated progression of carotid atherosclerosis, particularly among men with early evidence of atherosclerosis, and that chronically high levels of hopelessness may be especially detrimental. Additional research is needed to identify the contributory pathways and/or mechanisms underlying these relationships.
Both Intracellular and Extracellular Vitamin C Inhibit Atherogenic Modification of LDL by Human Vascular Endothelial Cells
Authors: Martin Antonio , Frei Balz
Published: June 2011

Abstract
Oxidative modification of LDL by vascular cells has been proposed as a mechanism by which LDL becomes atherogenic. Antioxidants that can prevent LDL oxidation may therefore act as antiatherogens. We used endothelial cells (ECs) from human aortas (HAECs), human saphenous veins (HSECs), and bovine aortas (BAECs) to investigate the role of intracellular and extracellular vitamin C (ascorbate) in EC-mediated LDL modification. Incubation of LDL (0.1 mg protein per milliliter) with confluent HAECs in Ham’s F-10 medium led to time-dependent modification of the lipoprotein. In contrast, incubation of LDL with HAECs in medium 199, which does not contain redox-active transition metal ions, did not lead to LDL modification. Both HAEC-mediated and cell-free LDL modifications in Ham’s F-10 medium were strongly inhibited in a time- and dose-dependent manner by physiological concentrations of ascorbate. Confluent HAECs cultured under conventional conditions contained very little intracellular ascorbate (<0.5 nmol/mg protein) but could be loaded with up to 20 nmol ascorbate per milligram protein in a time- and concentration-dependent manner. Ascorbate-loaded HAECs exhibited a lower capacity to modify LDL than did non–ascorbate-loaded control cells. When LDL was incubated with HSECs instead of HAECs, similar time- and concentration-dependent inhibitory effects on LDL modification of intracellular and extracellular ascorbate were observed. In contrast to human ECs, BAECs did not take up vitamin C and therefore only coincubation but not preincubation with ascorbate inhibited BAEC-mediated LDL modification. Our data show that enrichment of human vascular ECs with vitamin C lowers their capacity to modify LDL. In addition, extracellular vitamin C strongly inhibits EC-mediated, metal ion–dependent atherogenic modification of LDL.
A General Strategy for Isolation of Endothelial Cells From Murine Tissues
Authors: Dong Qiang Gang , Bernasconi Sergio , Lostaglio Susan , Wainstok De Calmanovici Rosa , Martin-Padura Ines , Breviario Ferruccio , Garlanda Cecilia , Ramponi Simona , Mantovani Alberto , Vecchi Annunciata
Published: June 2011

AbstractA rapid, reproducible method for the isolation of murine endothelial cells (ECs) has been developed. Murine ECs were highly enriched by collagenase digestion of mechanically minced lung and subcutaneous sponge implants followed by specific selection with rat anti-mouse CD31 (ie, PECAM-1) monoclonal antibody–coated magnetic beads (Dynabeads). Pure EC populations were isolated from primary cultures by a second cycle of immunomagnetic selection. The cells from the lung were then cloned by a limiting-dilution method to exclude the possibility of nonendothelial cell contamination. Of the 300 cells plated, 29 clones (≈10%) were obtained. The clones were positive for CD31 as measured by flow cytometry, and one clone from the lungs (1G11) and the cells from sponge implants (designated as SIECs) were then subjected to subsequent culture in vitro for 40 and 30 passages (up to 5 months), respectively. Characterization was performed on cells between passage 3 and 10. Both cell types formed contact-inhibited monolayers on gelatin and capillary-like “tubes” on Matrigel. However, 1G11 cells exhibited a “cobblestone” morphology, whereas SIECs had a fibroblast-like appearance at confluence. By flow cytometry and enzyme-linked immunosorbent assay, these cells constitutively expressed CD31, VE-cadherin (cadherin-5), CD34, ICAM-1, VCAM-1, and P-selectin. After stimulation with 30 ng/mL of tumor necrosis factor-α, the cells became positive for E-selectin (at 4 hours poststimulation) and the expression of ICAM-1, VCAM-1, and P-selectin was upregulated (after 24 hours of stimulation). The presence of VE-cadherin in 1G11 cells and SIECs was confirmed by fluorescence microscopy and Northern blot analysis. The phenotype and morphology of both cell types were stable during 5 months of culture, and there was no evidence of overgrowth by contaminating cells. Taken together, the approach outlined herein may provide a general strategy for the isolation and culture of ECs from a variety of murine tissues. The general strategy outlined here is simple, effective, and flexible, allowing the inclusion of further positive or negative selection steps.
Evidence That Apolipoprotein A-I
Milano
Has Reduced Capacity, Compared With Wild-Type Apolipoprotein A-I, to Recruit Membrane Cholesterol
Authors: Bielicki John K. , McCall Mark R. , Stoltzfus Lori J. , Ravandi Amir , Kuksis Arnis , Rubin Edward M. , Forte Trudy M.
Published: June 2011

Abstract
Human carriers of apolipoprotein (apo) A-I
Milano
are heterozygous for an Arg
173
→Cys substitution in the apoA-I primary sequence; despite severe reductions in HDL cholesterol concentrations, affected individuals do not develop coronary heart disease, suggesting that apoA-I
Milano
may possess antiatherogenic properties. As the beneficial effects of wild-type apoA-I are linked to its role in HDL cholesterol transport, we examined the capacity of apoA-I
Milano
to recruit cell cholesterol and activate lecithin:cholesterol acyltransferase (LCAT) (two key events in the antiatherogenic reverse cholesterol transport pathway). ApoA-I
Milano
and wild-type apoA-I were expressed in Chinese hamster ovary cells, and their ability to recruit membrane phospholipid and cholesterol for the assembly of nascent HDL was compared. Both clonal cell lines exhibited similar levels of apolipoprotein accumulation in serum-free medium (≈2 μg/mg cell protein per 24 hours), and 15% of each apolipoprotein was associated with membrane lipids to form nascent HDL (
d
=1.063 to 1.21 g/mL). SDS-PAGE showed that a majority (66±12%) of the lipidated apoA-I
Milano
was in the homodimer form. Compositional analyses revealed that apoA-I
Milano
nascent HDL had a significantly lower (
P
<.001) unesterified cholesterol/phospholipid mole ratio (0.47±0.10) than wild-type apoA-I complexes (1.29±0.14), indicating that apoA-I
Milano
had a reduced capacity to recruit cell cholesterol. In addition to the reduced unesterified cholesterol/phospholipid ratio, apoA-I
Milano
nascent HDL consisted mostly of small 7.4-nm particles compared with wild-type apoA-I, in which 11- and 9-nm particles predominated. Despite these changes in nascent HDL particle size and composition, apoA-I
Milano
activated LCAT normally. We conclude that, even though apoA-I
Milano
is a normal activator of LCAT, it is less efficient than wild-type apoA-I in recruiting cell cholesterol, suggesting that the putative antiatherogenic properties attributed to apoA-I
Milano
may be unrelated to the initial stages of reverse cholesterol transport.
Plasma Lipoprotein (a) Levels in Men and Women Consuming Diets Enriched in Saturated,
Cis
-, or
Trans
-Monounsaturated Fatty Acids
Authors: Clevidence Beverly A. , Judd Joseph T. , Schaefer Ernst J. , Jenner Jennifer L. , Lichtenstein Alice H. , Muesing Richard A. , Wittes Janet , Sunkin Matthew E.
Published: June 2011

Abstract
Studies that have shown adverse effects of
trans
-unsaturated fatty acids on plasma lipoprotein (a) [Lp(a)] levels have used levels of
trans
-fatty acid that are higher than those in the average U.S. diet. This study was conducted to clarify the effects on Lp(a) of
trans
-fatty acids levels commonly found in U.S. diets. Lp(a) levels were measured in a double-blind study of 29 men and 29 women who ate 4 controlled diets in random order for 6 weeks each. Fatty acids represented 39% to 40% of energy. The diets were: (1) Oleic (16.7% of energy as oleic acid); (2) Moderate
trans
(3.8% of energy as
trans
-monoenes, approximately the
trans
content of the U.S. diet); (3) High
trans
(6.6% of energy as
trans
-monoenes); (4) Saturated (16.2% of energy as lauric plus myristic plus palmitic acids). The Saturated diet lowered Lp(a) levels significantly (by 8% to 11%). Compared to the Oleic diet, the
trans
diets had no adverse effect on Lp(a) levels when all subjects were considered collectively. A subset with initially high levels of Lp(a) (≥30 mg/dL), however, responded to the High
trans
diet with a slight (5%) increase in Lp(a) levels relative to the Oleic and Moderate
trans
diets. Thus, in amounts commonly found in the typical U.S. diet, saturated fatty acids consistently decrease Lp(a) concentrations. The adverse effects of replacing
cis
- with
trans
-fatty acids are only suggestive and are restricted to high
trans
intakes in subjects with high Lp(a) levels.
Importance of Estrogen Receptors in Hepatic LDL Receptor Regulation
Authors: Parini Paolo , Angelin Bo , Rudling Mats
Published: June 2011

Abstract
Treatment with pharmacological doses of estrogen is the most potent way to stimulate hepatic LDL receptor expression in vivo. The mechanism for this effect is unclear, in part because of difficulties in inducing this stimulation in vitro. A fundamental question, whether estrogen receptors (ERs) mediate this stimulation, has not been addressed. The aim of the current study was to determine the involvement of ERs in the estrogen-induced stimulation of LDL receptors. Treatment of rats with high doses of ethynylestradiol for 7 days increased the hepatic LDL receptor protein and mRNA levels four- and threefold, respectively. LDL receptor stimulation in estrogen-treated rats was not due to their reduced food intake because hepatic LDL receptor expression did not increase in rats fasted for 72 hours. Treatment with antiestrogen (tamoxifen or clomiphene) abolished the LDL receptor stimulatory effect of ethynylestradiol at both the protein and mRNA levels. Antiestrogen alone had no effect on hepatic LDL receptor expression and did not influence the strong resistance to dietary cholesterol normally present in rats. It is concluded that ERs are critically involved in the induction of hepatic LDL receptor expression by ethynylestradiol. The known role of growth hormone for the expression of hepatic ERs may therefore play a role in the modulation of the effects of estrogen on cholesterol metabolism and hepatic LDL receptors in the rat.
Lipoprotein-like Phospholipid Particles Inhibit the Smooth Muscle Cell Cytotoxicity of Lysophosphatidycholine and Platelet-Activating Factor
Authors: Nilsson Jan , Dahlgren Britt , Ares Mikko , Westman Jan , Nilsson Anna Hultgårdh , Cercek Bojan , Shah Prediman K.
Published: June 2011

Abstract
—Oxidation of LDL is associated with degradation of phosphatidylcholine into platelet-activating factor (PAF)–like phospholipids and lysophosphatidylcholine (LPC). Exposure of cultured human smooth muscle cells to PAF and LPC in a concentration of 25 μmol/L was found to result in complete cell death, as assessed by the MTT cytotoxicity assay and cell counting. Addition of 50 μg/mL apolipoprotein A-I– and apolipoprotein A-I
Milano
–containing phospholipid particles completely inhibited this cytotoxicity. Phospholipid complexes alone were almost as effective, whereas free apolipoprotein A-I
Milano
and albumin were without effect, suggesting that the effect was phospholipid dependent. Experiments using [
14
C]LPC demonstrated that apolipoprotein A-I– and apolipoprotein A-I
Milano
–containing phospholipid particles effectively bind LPC. The results show that HDL-like phospholipid particles effectively inhibit the toxic effect of phospholipids and other lipid-soluble factors. The ability of HDL to inhibit the proinflammatory and toxic effects of phospholipids generated during oxidation of LDL may be responsible for part of the antiatherogenic properties of HDL.
Preferential Cholesteryl Ester Acceptors Among Triglyceride-Rich Lipoproteins During Alimentary Lipemia in Normolipidemic Subjects
Authors: Lassel Taous S. , Guérin Maryse , Auboiron Silvy , Chapman M. John , Guy-Grand Bernard
Published: June 2011

Abstract
—Triglyceride-rich lipoproteins (TRLs), namely chylomicrons (CMs), VLDL, and their remnants, are implicated in the atherogenic features of postprandial lipemia. In human plasma, cholesteryl ester transfer protein (CETP) mediates the heteroexchange of neutral lipids, ie, triglycerides (TG) and cholesteryl esters (CE), between distinct subpopulations of apoB- and of apoAI-containing lipoproteins. In fasting normolipidemic plasma, CETP plays an antiatherogenic role by promoting preferential CE redistribution from HDL to LDL particles of intermediate subclass with optimal binding affinity for the cellular LDL receptor. While the relative proportions and chemical compositions of donor and acceptor lipoproteins are known to influence CETP activity, elevated levels of TRL present during alimentary lipemia have been proposed to be associated with enhanced CETP activity. To identify the preferential CE acceptor particles among postprandial TRL subfractions, we investigated the effects of a typical Western meal (1200 kcal, 14% protein; 38% carbohydrate; and 48% fat, monounsaturated/polyunsaturated ratio 4:1) on the rates of postprandial CE transfer from HDL to apoB-containing lipoproteins in normolipidemic subjects (n=13). Two hours postprandially, plasma levels of TRL were significantly elevated (140 versus 51 mg/dL at baseline,
P
=.0001). Total rates of CE transferred (88±7 μg · h
−1
· mL
−1
) from HDL to apoB-containing lipoproteins were not significantly modified by alimentary lipemia over a period of 8 hours. Quantitatively, LDL accepted 64±5 μg CE per hour per milliliter plasma from HDL, whereas CM (Sf>400), VLDL1 (Sf 60 to 400), VLDL2 (Sf 20 to 60), and IDL (Sf 12 to 20) accepted 5±3, 16±3, 1.4±0.3, and 1.5±0.2, respectively. Quantitatively, VLDL1 was the major CE acceptor among TRLs (
P
=.0001); thus, VLDL1, but not CMs, represented the major CE acceptor among TRLs. Qualitatively however, VLDL2 and IDL displayed a higher capacity to accept CE from HDL (51.6±4.1 and 46.3±2.8 μg CE transferred per hour per milligram lipoprotein, respectively;
P
<.005) compared with CM, VLDL1, and LDL (12.6±2.8, 34.7±4.2, and 22.7±2.0 μg CE transferred per hour per milligram lipoprotein, respectively). In conclusion, elevated postprandial TRL levels are not associated with enhanced total CE transfer to these particles. Furthermore, the qualitative features of postprandial CE transfer from HDL to CM and VLDL1 were not related to the relative TG content of these particles. The CETP-facilitated enrichment of VLDL1 in CE therefore identifies them as potentially atherogenic particles during the postprandial phase.
Early Atherosclerotic Lesions Spiraling Through the Femoral Artery
Authors: Wensing Peter J. W. , Meiss Louis , Mali Willem P. T. M. , Hillen Berend
Published: June 2011

Abstract
—Atherosclerosis is common in the adductor hiatus region. The aim of this study was to evaluate atherosclerosis in relation to themorphological structure of the femoropopliteal region. Two anatomic features are thought to play an important role in the origin of these lesions: (1) curvature of the vessel, which may lead to unfavorable local hemodynamic factors that change during leg flexion; and (2) abrupt changes in stiffness of surrounding tissues of the vessel. The distal part of 23 postmortem femoral arteries were investigated. Cross sections were obtained every 1 mm over a length of 100 mm. For each cross section, lesion thickness was measured at 12 points along the circumference of the vessel. No apparent relation was found between surrounding structures of the femoral artery and location of atherosclerotic lesions. Three-dimensional reconstructions showed that atherosclerotic lesions were spiraling through the artery in 18 of 23 cases. Spiraling atherosclerotic lesions may be consistent with expected flow patterns in this part of the femoral artery.