bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and Function of a Recombinant PDGF B Gene in Porcine Arteries
Authors: Pompili Vincent J. , Gordon David , San Hong , Yang Zhiyong , Muller David W.M. , Nabel Gary J. , Nabel Elizabeth G.
Published: June 2011

AbstractPlatelet-derived growth factor (PDGF) B is a mitogen and chemoattractant for smooth muscle cells in vitro, and expression of a recombinant PDGF B gene in porcine arteries stimulates intimal thickening. To define the mechanisms by which PDGF B gene expression induces intimal thickening in vivo, we examined its effects on smooth muscle cell proliferation and migration, extracellular matrix synthesis, and inflammatory cell infiltration in intimal lesions of pig arteries after direct gene transfer of a recombinant PDGF B gene. PDGF B gene expression was associated with rapid formation of an intima, including 3- to 10-fold increases in intimal thickness and intima-to-media area ratio 4 to 21 days after gene transfer compared with control transfected arteries. Intimal smooth muscle cell proliferation was detected at 2 days, peaked at 7 days (P<.01), and declined by 14 days, although the total number of intimal nuclei progressively increased to 21 days (P<.01). Calculations of expected-to-observed ratios of intimal cells, based on BrdC proliferation indexes, demonstrated that the increases in intimal cell number on days 2 through 7 could not be accounted for by proliferation alone, suggesting that recombinant PDGF BB acts to stimulate cell proliferation and migration of smooth muscle cells into the intima. Extracellular matrix deposition and procollagen synthesis were observed after 7 days (P<.01) and were associated with a decline in cell density in the intima, suggesting that extracellular matrix synthesis may contribute to progressive intimal thickening in response to PDGF B gene expression. There was minimal accumulation of inflammatory cells, including macrophages and CD3(+) lymphocytes, in transfected arteries. These data suggest that PDGF B gene expression promotes intimal expansion by both proliferation and migration of smooth muscle cells followed by synthesis of extracellular matrix and therefore acts through several mechanisms to play a role in the pathogenesis of intimal lesions in vivo.
Sodium Butyrate Inhibits Platelet-Derived Growth Factor–Induced Proliferation of Vascular Smooth Muscle Cells
Authors: Ranganna Kasturi , Joshi Trupti , Yatsu Frank M.
Published: June 2011

Abstract
Sodium butyrate (SB), a naturally occurring short-chain fatty acid, was investigated for its therapeutic value as an antiproliferative agent for vascular smooth muscle cells (SMCs). At 5-mmol/L concentration, SB had no significant effect on rat SMC proliferation. However, at the same concentration, SB inhibited platelet-derived growth factor (PDGF)-AA–, -AB–, and -BB–induced proliferation of SMCs. Exposure of SMCs to PDGF-BB resulted in activation of receptor intrinsic tyrosine kinase activity and autophosphorylation of β-PDGF–receptor (β-PDGFR). The activated β-PDGFR physically associated and phosphorylated signaling molecules such as
ras
-GTPase activating protein (GAP) and phospholipase Cγ (PLCγ). SB, in the absence of PDGF-BB, caused neither β-PDGFR tyrosine phosphorylation nor phosphorylation and association of GAP and PLCγ with β-PDGFR. PDGF-BB–enhanced activation of receptor intrinsic tyrosine kinase activity and autophosphorylation of tyrosine residues of β-PDGFR were unaffected by SB irrespective of whether SMCs were preincubated with SB before exposure to PDGF-BB plus SB or incubated concomitantly with PDGF-BB plus SB. Likewise, phosphorylation and association of GAP and PLCγ with PDGF-BB–activated β-PDGFR were unaffected. In addition, SB did not block PDGF-BB–stimulated, PLCγ-mediated production of inositol triphosphate. Similarly, PDGF-BB–induced β-PDGFR degradation was unaffected when SMCs were exposed to PDGF-BB plus SB, and SB by itself had no influence on β-PDGFR degradation. Unlike β-PDGFR kinase activity, mitogen-activated protein kinase (MAP-kinase) activity was stimulated by SB by about 2.7-fold. Exposure of SMCs to PDGF-BB caused an ≈11.4-fold increase in MAP-kinase activity and this increase in activity was not significantly affected when cells were coincubated with PDGF-BB and SB (10.3-fold). However, pretreatment of SMCs with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB abolished most of the PDGF-BB–induced MAP-kinase activity (4.6-fold). Transcription of growth response genes such as c-
fos
, c-
jun
, and c-
myc
were induced by PDGF-BB, and their induction was suppressed, particularly c-
myc
, by incubating SMCs with PDGF-BB plus SB. Similarly, preincubation of cells with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB diminished PDGF-BB–induced transcription of c-
fos
, c-
jun
, and c-
myc
. However, SB by itself had no significant effect on c-
fos
, c-
jun
, and c-
myc
transcription. Our data suggest that the inhibition of PDGF-BB–induced proliferation of SMCs by SB involves MAP-kinase–regulated events as well as transcription of growth-response genes.
Effects of Alcohol Consumption on Lipoproteins of Premenopausal Women
Authors: Clevidence Beverly A. , Reichman Marsha E. , Judd Joseph T. , Muesing Richard A. , Schatzkin Arthur , Schaefer Ernst J. , Li Zhengling , Jenner Jennifer , Brown Charles C. , Sunkin Matthew , Campbell William S. , Taylor Philip R.
Published: June 2011

Abstract
A substantial portion of American women consume alcohol, but controlled studies of alcohol-induced changes in lipoproteins of women are rare. In this study, the effects of alcohol consumption (equivalent to two drinks per day) on the lipoprotein profiles of 34 premenopausal women were measured while controlling subjects’ diet and various other potentially confounding variables including phase of the menstrual cycle. Alcohol and no-alcohol treatments were administered in a crossover design, and blood samples were obtained during the early follicular phase of the third month of treatment. With alcohol, HDL cholesterol levels increased 10%, LDL levels decreased 8%, and levels of lipoprotein(a) were unchanged. The increase in HDL cholesterol was due to an increase in both HDL
2
and HDL
3
, and the overall size of HDL particles was increased. HDL particles containing apolipoprotein (apo) A-I and apoA-II as well as those containing apoA-I but no apoA-II were elevated in response to alcohol. Although these observations are limited to a single phase of the menstrual cycle, the alcohol-induced changes in lipoproteins are consistent with changes that are thought to confer protection against coronary heart disease.
Cholesterol Absorption and Metabolism and LDL Kinetics in Healthy Men With Different Apoprotein E Phenotypes and Apoprotein BXbaI and LDL ReceptorPvuII Genotypes
Authors: Gylling Helena , Kontula Kimmo , Miettinen Tatu A.
Published: June 2011

AbstractApoprotein (apo) E, apoBXbaI, and LDL receptor genePvuII polymorphisms are associated with LDL cholesterol level, but little is known about cholesterol and LDL metabolism in subjects with the latter two genetic polymorphisms alone or in combination with different apoE phenotypes. We studied cholesterol absorption efficiency, cholesterol and bile acid synthesis, and LDL apoB kinetics in 52 healthy men and related the metabolic results to the apoBXbaI and LDL receptorPvuII restriction fragment length polymorphism (RFLP) and apoE phenotypes. New findings were as follows. ApoBXbaI polymorphism was not associated with the metabolic variables of cholesterol, but LDL receptorPvuII RFLP was associated with fractional catabolic rate for LDL apoB, cholesterol absorption, and cholesterol and bile acid synthesis. ApoE polymorphism exerted the most powerful effect on the LDL cholesterol concentration, so that the apoE2 subjects had the lowest LDL cholesterol and apoB levels and cholesterol absorption, and the highest fractional catabolic rate and bile acid and cholesterol synthesis compared with the apoE3 or especially apoE4 phenotypes in different genetic combinations. In multiple stepwise regression analysis with LDL cholesterol as the dependent and the genetic and metabolic parameters as the independent variables, 47.0% (n=35,P<.001) of the variability of LDL cholesterol was explained by the apoE polymorphism, 7.1% (P<.05) by the LDL receptorPvuII RFLP, and 11.3% (P<.01) by bile acid synthesis, while the contribution of the apoBXbaI RFLP was nonsignificant. In conclusion, LDL cholesterol level was lowest in subjects with the ε2 allele, irrespective of theirXbaI orPvuII genotypes; this was due to lower cholesterol absorption efficiency, more effective cholesterol and bile acid synthesis, and more efficient fractional catabolic rate in carriers of the ε2 allele when compared with the other genetic subgroups.
Genotypic Variation in the Promoter Region of the Protein C Gene Is Associated With Plasma Protein C Levels and Thrombotic Risk
Authors: Spek C. Arnold , Koster Ted , Rosendaal Frits R. , Bertina Rogier M. , Reitsma Pieter H.
Published: June 2011

Abstract
Protein C is a vitamin K–dependent zymogen of a serine protease that inhibits blood coagulation by proteolytic inactivation of factors Va and VIIIa. Individuals with protein C deficiency are at risk for thrombophlebitis, deep-vein thrombosis, and pulmonary embolism. Genetic analysis of a number of randomly chosen healthy individuals revealed three polymorphisms, C/T at −654, A/G at −641, and A/T at −476, in the protein C promoter region. To investigate whether these genetic variations associate with the plasma protein C level, we determined the genotype for the three polymorphisms and measured plasma protein C levels in 240 individuals not deficient in protein C. The mean protein C level of these individuals was 103%. Interestingly, individuals with the homozygous CGT genotype (n=40) had a mean protein C level of 94%, whereas individuals with a homozygous TAA genotype (n=28) had a mean protein C level of 116%. This difference in mean protein C levels between the CGT and TAA groups (
P
<.001) could not be explained by environmental factors known to influence protein C levels in the normal population. Plasma factor II and factor X levels did not differ between the two groups, which makes a difference in liver function an unlikely cause. Finally, we tested whether the genotype associated with lower protein C levels is associated with higher thrombotic risks. This analysis showed that compared with the genetic variant associated with higher protein C levels (TT/AA/AA), the genetic variant associated with lower protein C levels (CC/GG/TT genotype) is indeed a risk factor for thrombosis (OR, 1.6; 95% confidence interval, 1.0 to 2.5).
Venous Thrombosis–Associated Inflammation and Attenuation With Neutralizing Antibodies to Cytokines and Adhesion Molecules
Authors: Wakefield Thomas W. , Strieter Robert M. , Wilke Carol A. , Kadell Amy M. , Wrobleski Shirley K. , Burdick Marie D. , Schmidt Regina , Kunkel Steven L. , Greenfield Lazar J.
Published: June 2011

AbstractThrombosis and inflammation are closely related. However, the response of the vein wall to venous thrombosis has been poorly documented. This study examines the hypothesis that venous thrombosis is associated with an inflammatory response in the vein wall. In a rat model of inferior vena caval thrombosis, vein wall was temporally examined for inflammation by assessment of histopathology, leukocyte morphometrics, and cytokine levels. Animals were killed 1 hour and 1, 3, and 6 days after thrombus induction. Our findings demonstrated an early (day 1) neutrophil infiltration into the vein wall followed by a later (days 3 and 6) monocyte/macrophage and lymphocyte response. Cytokines were elevated only under conditions of venous thrombosis. Levels of epithelial neutrophil activating protein–78 (ENA-78), tumor necrosis factor–α (TNF), interleukin-6, and JE/monocyte chemoattractant protein–1 (JE/MCP-1) increased over the 6-day period, while macrophage inflammatory protein-1α (MIP-1α) peaked at day 3 after thrombus induction. Additionally, rats were passively immunized with neutralizing antibodies to TNF, ENA-78, MIP-1α, JE/MCP-1, intercellular adhesion molecule–1 (ICAM-1), and CD18 compared with control antibodies. The most effective antibody early after thrombus induction for attenuating vein wall neutrophil extravasation was anti-TNF (P<.01). The monocyte/macrophage extravasation was inhibited most by anti–ICAM-1 followed by anti-TNF (P<.01). These findings demonstrate that venous thrombosis is associated with significant vein wall inflammation that is partially inhibited by neutralizing antibodies to cytokines and adhesion molecules.
Vitamin E and Fatty Acid Intervention Does Not Attenuate the Progression of Atherosclerosis in Watanabe Heritable Hyperlipidemic Rabbits
Authors: Kleinveld Henne A. , Hak-Lemmers Heidi L.M. , Hectors Magda P.C. , de Fouw Nanneke J. , Demacker Pierre N.M. , Stalenhoef Anton F.H.
Published: June 2011

Abstract
We investigated the effect of different interventions on aortic atherosclerosis in Watanabe rabbits. Four groups of rabbits were fed either an oleic acid–enriched diet (80% of total fat intake) with or without vitamin E supplementation (250 IU/kg) or a diet enriched in linoleic acid with or without vitamin E supplementation for 6 months. At the start of the study, plasma cholesterol concentration was 21.4±3.6 mmol/L (n=32). The diets did not influence the mean plasma lipids and lipoprotein concentrations except for HDL cholesterol, which was increased more on the oleic acid–enriched diets than on the linoleic acid–enriched diets. Vitamin E levels in plasma and LDL were increased on the oleic acid diet and reduced on the linoleic acid diet. On the latter diet, supplementation of vitamin E was quantitatively less effective in raising plasma or LDL vitamin E levels. The susceptibility of LDL to oxidation was determined in vitro. Both oleic acid–enriched diets increased the lag time by 140% from baseline. The linoleic acid diet supplemented with vitamin E increased lag time by 59%. Linoleic acid alone, however, decreased the lag time by 30%. Similar but inverse effects were seen on LDL oxidation rate. Thus, intervention protected LDL to oxidation in the following order: oleic acid plus vitamin E>oleic acid>linoleic acid plus vitamin E>linoleic acid. Despite the differences in LDL oxidizability induced by the four experimental diets, assessment of aortic atherosclerosis at the end of the 6-month dietary study period revealed no differences among the four study groups. These results suggest that a decrease in the oxidative susceptibility in vitro alone is not sufficient to attenuate atherogenesis when cholesterol levels remain markedly elevated.
Persistence of Low HDL-C Levels After Weight Reduction in Older Men With Small LDL Particles
Authors: Katzel Leslie I. , Coon Patricia J. , Rogus Ellen , Krauss Ronald M. , Goldberg Andrew P.
Published: June 2011

Abstract
LDL subclass pattern B is characterized by a predominance of small LDL particles (LDL peak particle size ≤255 Å) and is associated with increased plasma triglyceride (TG) and reduced HDL cholesterol (HDL-C) concentrations. This study compared the effect of weight loss on lipoprotein and glucose metabolism in 15 healthy, obese (body mass index [BMI], 30.9±2.4 kg/m
2
), older (60±9 years) men with LDL pattern B and in 25 men of comparable age and BMI with LDL pattern A (LDL peak particle size ≥260 Å). At baseline, men with LDL pattern B had higher TG and lower apolipoprotein (apo) A-I, HDL-C, and HDL
2
-C levels (
P
<.001) than men with LDL pattern A, while the total cholesterol and LDL cholesterol levels and fasting and 2-hour postprandial glucose and insulin levels did not differ between groups. With weight loss (10.1±3.6 kg) there were significant decreases in 2-hour postprandial glucose and insulin levels in men with LDL patterns B and A (
P
<.05). However, the change in plasma TG, HDL-C, HDL
2
-C, and apoA-I levels with weight loss differed between groups. In men with LDL pattern A, plasma TG levels decreased by 15% (
P
<.001) compared with a 34% (
P
<.001) decrease in LDL pattern B (two-factor ANOVA,
P
<.01). Plasma HDL-C concentrations increased by 0.16 mmol/L (
P
<.001) in the men with LDL pattern A but by only 0.07 mmol/L in the men with LDL pattern B (two-factor ANOVA,
P
<.05). After weight loss, only 5 of the 15 men with LDL pattern B had HDL-C levels above 0.90 mmol/L (35 mg/dL), whereas 22 of 25 men with LDL pattern A had HDL-C levels above 0.90 mmol/L (χ
2
=18,
P
<.0001). Furthermore, with weight loss, 11 of the 15 men with LDL pattern B increased their LDL peak particle diameter; 7 converted to intermediate LDL pattern, and 4 converted to LDL pattern A. By comparison, there were no significant changes in weight, lipoprotein, or apolipoprotein concentrations at 1-year follow-up in 12 metabolic control subjects. Thus, despite significant reductions in weight and body fat and concomitant decreases in plasma TG and insulin levels, HDL-C and HDL
2
subspecies levels remain low in men with LDL pattern B.
Phenotypic Correction of Hypercholesterolemia in ApoE-Deficient Mice by Adenovirus-Mediated In Vivo Gene Transfer
Authors: Stevenson Susan C. , Marshall-Neff Jennifer , Teng Babie , Lee Cadir B. , Roy Soumitra , McClelland Alan
Published: June 2011

Abstract
To investigate the potential use of apoE in gene therapy of hyperlipidemias, an adenoviral vector was constructed that contained the human apoE3 cDNA under the control of the RSV promoter (Av1RE). Transduction of HepG2 cells resulted in the overexpression of human apoE secreted into the culture medium. Intravenous injection of 5×10
11
Av1RE vector particles into apoE-deficient mice resulted in expression of human apoE3 in mouse plasma at levels of 1.2±0.4 μg/mL (mean±SEM, n=5) 7 days after injection. Mice injected with the control vector Av1Lacz4 did not express detectable levels of human apoE. Average plasma cholesterol concentrations were reduced approximately eightfold from 737.5±118 mg/dL (mean±SEM, n=6) to 98.2±4.4 mg/dL (mean±SEM, n=5) and were unaffected in the control vector group. Expression of human apoE resulted in a shift in the plasma lipoprotein distribution from primarily VLDL and LDL in the control mice to predominantly HDL in the Av1RE-treated group. Western blot analysis of fast protein liquid chromatography–fractionated mouse plasma showed that the human apoE protein was associated with VLDL, LDL, and HDL. Correction of the hyperlipidemic condition found in the apoE-knockout mouse strain by direct in vivo gene transfer establishes the potential of this approach for treatment of hyperlipidemia caused by apoE deficiency or malfunction in human disease.
Synthetic Analogues of the Antithrombin III– Binding Pentasaccharide Sequence of Heparin
Authors: van Amsterdam Ronald G.M. , Vogel Gerard M.T. , Visser Arie , Kop Wim J. , Buiting Marc T. , Meuleman Dirk G.
Published: June 2011

AbstractThe synthetic pentasaccharide Org 31540/SR 90107A represents the antithrombin III (ATIII) binding region of heparin and accelerates the ATIII-mediated inhibition of coagulation factor Xa. This compound and 15 structural analogues with ATIII binding constants (Kd) ranging from 2.7 to 2600 nmol/L were compared for their plasma elimination in rats as measured from their factor Xa inhibiting activity. After administration of a low dose (100 nmol/kg body wt IV), each pentasaccharide showed a characteristic plasma half-life varying from a minimum of 0.3 hour for pentasaccharides with low affinity for ATIII to 10.9 hours for pentasaccharides with high affinity for the protein. The latter value was close to the half-life measured for radioiodinated rat ATIII (11.8 hours). We hypothesized that the elimination half-life of pentasaccharides is markedly extended by ATIII binding, of which the extent is governed by theKdof the complex. The following observations support this hypothesis. The low-dose, low-affinity pentasaccharides were almost fully recovered in the urine without having lost anti–factor Xa activity, whereas compounds with high ATIII binding affinity were only partly recovered in the urine. With a high dose (500 nmol/kg body wt), a rapid plasma clearance of pentasaccharide was observed until a concentration similar to that of endogenous ATIII was reached, in accordance with their expected 1:1 stoichiometric interaction. The elimination half-life was similar to that of the low dose. The relation betweenKdvalues and plasma half-lives could be explained by assuming rapid clearance of free and coclearance of ATIII-bound pentasaccharide with the protein. We applied the plasma ATIII concentration (3.5 μmol/L), the half-life of ATIII (11.8 hours), the half-life of unbound pentasaccharides (<10 minutes), and theKdvalues and concluded that highly specific binding to ATIII in the circulation governs the presented straightforward pharmacological profile for the pentasaccharides.
Selective Retention of VLDL, IDL, and LDL in the Arterial Intima of Genetically Hyperlipidemic Rabbits In Vivo
Authors: Nordestgaard Børge G. , Wootton Richard , Lewis Barry
Published: June 2011

AbstractTo explore possible mechanisms whereby the triglyceride-rich lipoproteins IDL and VLDL may promote atherosclerosis, fractional loss of these lipoproteins from the intima–inner media was measured in vivo in genetically hyperlipidemic rabbits of the St Thomas’s Hospital strain and compared with the fractional loss of LDL, HDL, and albumin. These rabbits exhibit elevated plasma levels of VLDL, IDL, and LDL. In each rabbit, two aliquots of the same macromolecule, one iodinated with125I and the other with131I, respectively, were injected intravenously on average 24 and 3 hours, respectively, before removal of the aortic intima–inner media. The fractional loss from the intima–inner media of newly entered macromolecules was then calculated. The average fractional losses for VLDL, IDL, LDL, HDL, and albumin in lesioned aortic arches were 0.1%/h (n=4), −0.2%/h (n=3), 1.8%/h (n=4), 11.4%/h (n=3), and 26.3%/h (n=1), respectively; in nonlesioned aortic arches fractional losses for IDL, LDL, HDL, and albumin were 1.7%/h (n=1), 0.6%/h (n=2), 14.6%/h (n=3), and 25.9%/h (n=3). In both lesioned and nonlesioned aortic arches, the logarithms of these fractional loss values were inversely and linearly dependent on the diameter of the macromolecules (R2=.57,P=.001 andR2=.84,P<.001), as determined from electron photomicrographs of negatively stained lipoproteins. These results suggest that after uptake into the arterial intima, VLDL and IDL as well as LDL are selectively retained in comparison with HDL and albumin.
Immunological Evidence for the Presence of Advanced Glycosylation End Products in Atherosclerotic Lesions of Euglycemic Rabbits
Authors: Palinski Wulf , Koschinsky Theodor , Butler Susan W. , Miller Elizabeth , Vlassara Helen , Cerami Anthony , Witztum Joseph L.
Published: June 2011

Abstract
Atherosclerosis is known to be accelerated in diabetic patients, but the mechanisms of this acceleration are poorly understood. Nonenzymatic glycosylation of long-lived proteins results in the formation of advanced glycosylation end products (AGEs), which are extensively cross-linked and could contribute to atherogenesis. Oxidative modification of LDL is also an important process in atherogenesis. In vitro evidence suggests that hyperglycemia may enhance lipid peroxidation, and conversely, that increased lipid peroxidation may enhance AGE formation. If such interactions occur in vivo, we hypothesized that AGE should be found in atherosclerotic lesions of euglycemic LDL receptor–deficient rabbits in areas rich in lipids and oxidized lipoproteins. To demonstrate the presence of AGEs, we developed antisera against a specific “model” compound of AGE, 2-furoyl-4(5)-(2-furanyl)-
1H
-imidazole (FFI) by using FFI–hexanoic acid (FFI-HA)–protein adducts as the antigen and against AGEs in general by using AGE-albumin as the antigen. Antisera generated with FFI-HA–protein adducts recognized FFI-HA alone as well as FFI-protein adducts. Native proteins or proteins conjugated with aldehydes formed during lipid peroxidation in vitro were not recognized by these antisera. Immunocytochemistry with both FFI-specific and AGE-specific antisera revealed the presence of these epitopes in atherosclerotic lesions of euglycemic LDL receptor–deficient rabbits but not in normal aortic tissues. AGE epitopes within atherosclerotic lesions were predominantly found in similar locations as epitopes generated during modification of the lipoproteins by oxidation, consistent with the hypothesized interactions between oxidation and glycosylation. Indirect evidence in support of the in vivo presence of FFI-like structures was also obtained by the observation that both diabetic and euglycemic human subjects contained autoantibodies that recognize FFI-protein adducts. Taken together, these data provide immunological evidence for the in vivo presence of FFI-like structures and other AGE-protein adducts in atherosclerotic lesions, even in euglycemic conditions.
The Lipoprotein Lipase
Hin
dIII Polymorphism Modulates Plasma Triglyceride Levels in Visceral Obesity
Authors: Vohl Marie-Claude , Lamarche Benoı̂t , Moorjani Sital , Prud’homme Denis , Nadeau André , Bouchard Claude , Lupien Paul-J. , Després Jean-Pierre
Published: June 2011

Abstract
The aim of this study was to investigate the potential interaction between the lipoprotein lipase (LPL)
Hin
dIII polymorphism and visceral adipose tissue (AT) accumulation in the modulation of triglyceride levels in visceral obesity. The LPL-
Hin
dIII genotype was determined by polymerase chain reaction in 52 men. Twenty-three subjects were heterozygous (+/−) and 28 were homozygous (+/+) for the presence of the restriction site. One subject who was homozygous for the − allele was excluded from analysis. Body mass index (BMI), fasting insulin level, and visceral AT area as measured by computed tomography were positively correlated with triglyceride levels only in subjects homozygous for the + allele. Furthermore, whereas these variables were negatively correlated with plasma HDL
2
cholesterol concentrations in the +/+ group, these associations were not found in +/− heterozygotes, with the exception of BMI. To further investigate the interaction of the LPL-
Hin
dIII polymorphism with visceral obesity and hyperinsulinemia, the two genotype groups were further subdivided on the basis of BMI (low versus high), fasting insulin level (low versus high), and visceral AT area (low versus high), and their lipoprotein profiles were compared. Elevated levels of abdominal visceral AT were significantly associated with increased triglyceride concentrations in +/+ homozygous men, suggesting that visceral obesity may lead to hypertriglyceridemia in the presence of the +/+ genotype. In the +/− group, variation in the amount of visceral AT was not associated with differences in triglyceride concentration. However, hypertriglyceridemia and an increased cholesterol-to–HDL cholesterol ratio were observed in the hyperinsulinemic state irrespective of LPL-
Hin
dIII genotype status. Finally, similar positive correlations were observed between visceral AT accumulation and plasma insulin level in the homozygous (+/+) and heterozygous (+/−) groups, suggesting that the hyperinsulinemic–insulin-resistant state that is frequently associated with visceral obesity is independent of LPL-
Hin
dIII genotype. These results suggest that the
Hin
dIII polymorphism may modulate the magnitude of the dyslipidemic state associated with visceral obesity.
Evidence That Meizothrombin Is an Intermediate Product in the Clotting of Whole Blood
Authors: Bovill Edwin G. , Tracy Russell P. , Hayes Timothy E. , Jenny Richard J. , Bhushan Francis H. , Mann Kenneth G.
Published: June 2011

Abstract
Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospholipid surface. The biological significance of this intermediate has recently been challenged by the apparent absence of meizothrombin during clotting of sodium citrate–anticoagulated plasma. We analyzed the formation of thrombin during coagulation of nonanticoagulated, unchilled, minimally manipulated whole blood in glass tubes. The process was stopped at 0, 3, 5, and 7 minutes by the addition of biotinylated peptidyl chloromethylketone active-site labeling reagents. Plasma/serum was separated by centrifugation, and labeled species were extracted by immunoadsorption with a polyclonal anti-prothrombin antibody. The purified prothrombin-derived species were separated by SDS–polyacrylamide gradient gel electrophoresis and visualized on a chemiluminescent avidin blot. Meizothrombin appeared as an intermediate product of this reaction and persisted with some increase through the 7-minute time point. We also observed incorporation of the active-site label into a species of lower molecular weight consistent with the B
1
chain of β- and/or γ-thrombin. These degraded forms of thrombin have not been previously demonstrated in a biologically relevant preparation. Our data clearly establish the generation of meizothrombin as an intermediate product of thrombin generation during whole-blood clotting. The data also represent the first experimental evidence for the generation of β- and γ-thrombin in a biologically relevant environment and time scale.
FH Afrikaner-3 LDL Receptor Mutation Results in Defective LDL Receptors and Causes a Mild Form of Familial Hypercholesterolemia
Authors: van Roggen J.F. Graadt , van der Westhuyzen D.R. , Coetzee G.A. , Marais A.D. , Steyn K. , Langenhoven E. , Kotze M.J.
Published: June 2011

Abstract
Three founder-related gene mutations (FH Afrikaner-1, -2, and -3) that affect the LDL receptor are responsible for 90% of the familial hypercholesterolemia (FH) in South African Afrikaners. Patients heterozygous for the FH Afrikaner-1 (FH1) mutation, which results in receptors having ≈20% of normal receptor activity, have significantly lower plasma cholesterol levels and milder clinical symptoms than heterozygotes with the FH Afrikaner-2 mutation, which completely abolishes LDL receptor activity. In this study we re-created the FH3 mutation (Asp
154
→Asn) in exon 4 by site-directed mutagenesis and analyzed the expression of the mutant receptors in Chinese hamster ovary cells. The mutation resulted in the formation of LDL receptors that are markedly defective in their ability to bind LDL, whereas binding of apoE-containing β-VLDL is less affected. The mutant receptors are poorly expressed on the cell surface as a result of significant degradation of receptor precursors. The plasma cholesterol levels of 31 FH3 heterozygotes were similar to FH1 heterozygotes but significantly lower than FH2 heterozygotes. The FH1 and FH3 heterozygotes also tended to be less severely affected clinically (by coronary heart disease and xanthomata) than FH2 patients. This study demonstrates that mutational heterogeneity in the LDL receptor gene influences the phenotypic expression of heterozygous FH and that severity of expression correlates with the activity of the LDL receptor measured in vitro. The results further indicate that knowledge of the specific mutation underlying FH in heterozygotes is valuable in determining the potential risk of premature atherosclerosis and should influence the clinical management of FH patients.
IgE-Dependent Generation of Foam Cells: An Immune Mechanism Involving Degranulation of Sensitized Mast Cells With Resultant Uptake of LDL by Macrophages
Authors: Ma Hua , Kovanen Petri T.
Published: June 2011

AbstractBecause a role has been suggested for IgE in cardiovascular diseases and for mast cells in cholesterol accumulation within the macrophages of atherosclerotic lesions, we examined mast cell–macrophage interactions in vitro by using rats with high serum levels of IgE antibodies. The rats were immunized with an antigen (ovalbumin) and adjuvant (Bordetella pertussisvaccine) to provoke synthesis of IgE and to sensitize their mast cells, ie, to allow the IgE to bind to the high-affinity IgE receptors on the mast cell surfaces. On addition of the ovalbumin to suspensions of mast cells isolated from the peritoneal cavity of the immunized rats, the mast cells responded by exocytosing their heparin-proteoglycan–containing granules. When IgE-bearing peritoneal mast cells were cocultured with peritoneal macrophages (also from the immunized rats) in a medium enriched in LDL, addition of ovalbumin to the incubation medium triggered a dose-dependent release of granules and a dose-dependent increase in the rate of LDL uptake by the macrophages. In contrast, ovalbumin had no effect on LDL uptake if the cultures contained only macrophages or if the mast cells and macrophages were from nonimmunized rats. Thus, the sequence of events leading to enhanced uptake of LDL by macrophages depended wholly on IgE-dependent degranulation of the sensitized mast cells. With the aid of gold-labeled LDL we demonstrated that the exocytosed mast cell granules had bound LDL particles and carried them into the macrophages, with subsequent formation of foam cells. The current series of experiments delineates a novel immunologic mechanism for the formation of macrophage foam cells and assigns a potentially atherogenic role to mast cell–bound IgE antibodies.
Multiple Genetic Determinants of Variation of Plasma Lipoproteins in Alberta Hutterites
Authors: Hegele Robert A. , Brunt J. Howard , Connelly Philip W.
Published: June 2011

Abstract
We hypothesized that variation of nine candidate genes in lipoprotein metabolism would be associated with variation in fasting plasma lipoprotein variables in 718 Alberta Hutterites, a genetic isolate. We measured plasma lipids, lipoproteins, and apolipoproteins and analyzed DNA for genotypes of apolipoprotein (apo) B (
APOB
), paraoxonase (
PON
), lipoprotein lipase (
LPL
), VLDL receptor (
VLDLR
), apo CIII (
APOC3
), LDL receptor–related protein (
LRP
), hepatic lipase (
HL
), LDL receptor (
LDLR
), and apo E (
APOE
). Using a multivariate analysis, we found that (1) genotypes of
APOB
,
PON
,
LPL
,
LDLR
, and
APOE
were significantly associated with variation of plasma apo B–related traits; (2) genotypes of
PON
,
LPL
, and
APOC3
were significantly associated with variation in plasma triglycerides; and (3) genotypes of
VLDLR
,
APOC3
,
LDLR
, and
APOE
were significantly associated with variation in plasma apo AI and HDL cholesterol. Regression analysis showed that between 3.2% and 7.8% of the total variation in plasma lipoproteins was accounted for by variation in the candidate genes tested. The observations demonstrate a modest but significant genetic component of variation in plasma lipoprotein levels that is due to the candidate genes studied in this normolipemic human genetic isolate.
In Vivo LDL Receptor and HMG-CoA Reductase Regulation in Human Lymphocytes and Its Alterations During Aging
Authors: Stulnig Thomas M. , Klocker Helmut , Harwood H. James , Jürgens Günther , Schönitzer Dieter , Jarosch Elmar , Huber Lukas A. , Amberger Albert , Wick Georg
Published: June 2011

Abstract
The LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase play primary roles in the regulation of cellular cholesterol metabolism. To investigate the transcriptional regulation of lipid metabolism under physiological conditions ex vivo and its alterations during aging, we analyzed both the activity and mRNA concentration of the LDL receptor and HMG-CoA reductase in freshly isolated lymphocytes from healthy young and elderly donors. Data from fluorescent reverse transcriptase–polymerase chain reaction indicated that not only plasma LDL but also plasma HDL downregulates lymphocyte LDL receptor mRNA. Downregulation by HDL was three times more effective than that by LDL and presumably involved specific HDL binding sites. There was coordinate regulation of HMG-CoA reductase mRNA with LDL receptor mRNA that was independent of plasma lipoprotein concentrations. Despite elevated plasma concentrations of LDL, lymphocytes from elderly donors paradoxically expressed increased levels of the LDL receptor (
P
=.030) and HMG-CoA reductase mRNA (
P
=.062). The age-related dysregulation of the LDL receptor was predominantly due to impaired downregulation by plasma LDL rather than by HDL. Thus, not only LDL but also HDL and age significantly influences the transcriptional regulation of the LDL receptor in extrahepatic cells in vivo.
Quantification of Plasminogen Activators and Their Inhibitors in the Aortic Vessel Wall in Relation to the Presence and Severity of Atherosclerotic Disease
Authors: Padró Teresa , Emeis Jef J. , Steins Martin , Schmid Kurt W. , Kienast Jochen
Published: June 2011

AbstractIncreased expression of plasminogen activator inhibitor–1 (PAI-1) has been demonstrated in the human atherosclerotic vessel wall and may contribute to the impaired plasma fibrinolytic capacity in patients at high risk of atherothrombotic events. In addition, the mural PA/plasmin system may have important pathobiologic functions during atherogenesis. We quantitatively analyzed PAs of the tissue type (TPA) and urokinase type (UPA), PAIs, and plasminogen in protein extracts from different layers of human aorta in relation to the presence and severity of atherosclerotic lesions. In comparison with normal control vessels, intimal and neointimal TPA concentrations were reduced in atherosclerotic aortas except in the necrotic core areas of advanced plaques, where TPA was mainly complexed to PAI-1 in extracellular matrix deposits. In the media, TPA antigen was higher in lesional segments and closely associated with smooth muscle cells. UPA antigen was increased in the intima of atherosclerotic lesions and colocalized with tissue-infiltrating macrophages and neointimal smooth muscle cells. By spectrophotometric assay, neither TPA nor UPA activity could be detected in intimal or medial extracts. PAI-1 concentrations increased significantly in the intima of atherosclerotic segments compared with adjacent uninvolved areas or control aortas. The immunohistochemical distribution of PAI-1 was similar to that observed for TPA. A large excess of PAI-1 over PA concentrations, particularly in the intimal layer, characterizes atherosclerotic lesions of the human aorta and suggests that PA action is locally confined and counterbalanced by enhanced PAI expression and accumulation.
Different Effects of Subclasses of HDL Containing ApoA-I but Not ApoA-II (LpA-I) on Cholesterol Esterification in Plasma and Net Cholesterol Efflux From Foam Cells
Authors: Ohta Takao , Saku Keijiro , Takata Kouki , Nakamura Rie , Ikeda Yoichiro , Matsuda Ichiro
Published: June 2011

Abstract
We investigated the effects of subclasses of plasma LpA-I (HDL containing apoA-I but not apoA-II) on cholesterol esterification in plasma and net cholesterol efflux from foam cells. LpA-I was composed of particles of three diameters: large (11.1 nm; Lg–LpA-I), medium (8.8 nm; Md–LpA-I), and small (7.7 nm; Sm–LpA-I). Plasma concentrations of LpA-I were positively correlated only with the level of Lg–LpA-I. Plasma concentrations of Lg–LpA-I were inversely correlated with the rate of cholesterol esterification in plasma and VLDL- and LDL-depleted plasma. Plasma concentrations of Md–LpA-I and Sm–LpA-I did not correlate with the rate of cholesterol esterification in plasma or VLDL- and LDL-depleted plasma. When macrophage foam cells were incubated with Md– and Sm–LpA-I, cellular cholesterol mass was reduced by approximately 70%. In contrast, the cellular cholesterol–reducing capacity of Lg–LpA-I was negligible. Lg–LpA-I inhibited net cholesterol removal from foam cells that was mediated by Md– and Sm–LpA-I and cholesteryl ester production with these particles. These results suggest that Md– and Sm–LpA-I may actively participate in cellular cholesterol removal and cholesterol esterification in plasma and HDL, while Lg–LpA-I may regulate these functions of Md– and Sm–LpA-I.
Modulation of Lipoprotein B Binding to the LDL Receptor by Exogenous Lipids and Apolipoproteins CI, CII, CIII, and E
Authors: Clavey V. , Lestavel-Delattre S. , Copin C. , Bard J. M. , Fruchart J. C.
Published: June 2011

Abstract
We have recently shown that apo B–containing lipoproteins isolated by immunoaffinity chromatography bind to the LDL receptor with an affinity dependent on their apo E or apo CIII content. However, these lipoproteins—LpB:E, LpB:CIII, and LpB:CIII:E—isolated from whole plasma have variable lipid and apolipoprotein contents, and it is difficult to consider each parameter separately, particularly because an increase in the apo CIII content is always associated with an increase in the content of other C apolipoproteins. Therefore, we used affinity-purified LpB free of other apolipoproteins. Lipid content of LpB was increased by incubation with a lipid emulsion, and this triglyceride-enriched LpB was named TG-LpB. Free apo CI, apo CII, apo CIII, and apo E were added to LpB and TG-LpB and their associations to the lipoprotein were assessed by gel filtration, nondenaturing electrophoresis, and immunoblotting. Molar ratios of 6 (apo E), 30 (apo CII), 20 (apo CIII), and 30 (apo CI) for 1 apo B were obtained. The association of apo CII to LpB and TG-LpB induced modifications to the LpB structure and a redistribution of lipids and apolipoproteins on the lipoprotein particles. The binding of these LpBs and TG-LpBs with and without added apo CI, CII, CIII, and E was tested at 4°C on the LDL receptors of HeLa cells. The increased content of lipids reduced TG-LpB binding to the LDL receptor. Addition of apo CIII to LpB decreased its affinity, although this decrease was lower than that observed with LpB:CIII prepared from whole plasma. Apo CIII almost completely abolished the interaction of TG-LpB with the receptor, indicating a synergistic effect of lipids and apo CIII. The apo CIII effect was specific and cannot be obtained with apo CI. With apo CII, an inhibitory effect can also be obtained but to a lesser extent than with apo CIII. At 37°C the C apolipoproteins decreased the catabolism of LpB and TG-LpB by the LDL receptor of fibroblasts. Addition of apo E to either LpB or TG-LpB had a small effect on the binding of the enriched lipoproteins at 4°C but markedly increased their catabolism at 37°C.
Immune Complexes of LDL Induce Atherogenic Responses in Human Monocytic Cells
Authors: Kiener Peter A. , Rankin Bruce M. , Davis Patricia M. , Yocum Sue A. , Warr Glenn A. , Grove Robert I.
Published: June 2011

AbstractThe ability of immune complexes of LDL or acetylated LDL (acLDL), together with antibodies to LDL, to induce a proatherogenic phenotype in human monocytic cells has been explored. Treatment of THP-1 monocytic cells or peripheral human monocytes with LDL immune complexes containing intact anti-LDL markedly enhanced the ability of these cells to subsequently bind and take up LDL, whereas aggregated LDL or LDL immune complexes prepared with F(ab′)2fragments of anti-LDL had no significant effect. Activation of THP-1 cells with intact LDL immune complexes also stimulated mRNA expression for the scavenger receptor. Additionally, activation of THP-1 cells with insoluble immune complexes of LDL or LDL stimulated generation of reactive oxygen intermediates that, in turn, could oxidize exogenous LDL. These results indicate that the binding of lipoprotein immune complexes to Fc receptors on monocytic cells activates a series of responses that could accelerate the initiation or progression of atherosclerosis.
Apo(a) Phenotypes and Lp(a) Concentrations in Offspring of Men With and Without Myocardial Infarction
Authors: Klausen Ib Christian , Beisiegel Ulrike , Menzel Hans-Jürgen , Rosseneu Maryvonne , Nicaud Viviane , Faergeman Ole
Published: June 2011

Abstract
In the European Atherosclerosis Research Study, genetic and environmental markers of risk of premature coronary heart disease were compared in offspring of men with and without myocardial infarction before the age of 55 years. Cases were 682 students with a paternal history of myocardial infarction, and control subjects were 1312 students without such a history. The students were enrolled in 14 universities in five European regions (Finland, Great Britain, and northern, middle, and southern Europe). Lipoprotein(a) [Lp(a)] concentrations were skewed towards lower concentrations in both cases (median, 7.3 mg/dL; 95% confidence interval, 6.3 to 8.1 mg/dL) and control subjects (median, 6.6 mg/dL; 95% confidence interval, 6.1 to 7.2 mg/dL) (
P
=.37). Significantly more northern European male cases than control subjects had Lp(a) levels exceeding 30 mg/dL (
P
=.040), but this did not pertain to females (
P
=.29), and overall, there was no difference between cases (16.5%) and control subjects (15.5%) in the frequency of Lp(a) concentrations above 30 mg/dL (
P
=.63). As expected, there was a significant (
P
<.01) inverse relationship between apo(a) molecular size and Lp(a) concentration. In Great Britain there was a significant difference in phenotype distribution between cases and control subjects (
P
=.035), due mainly to a high frequency of the apo(a) S2 isoform in cases. A similar but statistically insignificant tendency was seen in northern Europeans. In the three other regions, however, the distribution of apo(a) phenotypes among cases and controls was similar, and in the study population overall, the distribution of apo(a) phenotypes did not differ significantly (
P
=.74) between cases and control subjects.
Determinants of Fibrin
d
-Dimer in the Edinburgh Artery Study
Authors: Lee Amanda J. , Fowkes F. Gerald R. , Lowe Gordon D. O. , Rumley Ann
Published: June 2011

Abstract
There is growing evidence that fibrin
d
-dimer is associated with coronary and peripheral atherosclerosis. Using data from the Edinburgh Artery Study, we examined the distribution of fibrin
d
-dimer in 1592 men and women 55 to 74 years old and assessed its relationship with a range of cardiovascular risk factors. Fibrin
d
-dimer levels were higher in women than in men (
P
≤.05) and increased with age (
P
≤.001). Current cigarette smokers had higher levels than ex-smokers, who, in turn, had higher levels than those who had never smoked. On multiple regression analyses with age and plasma fibrinogen as covariates, only lifetime smoking in men and systolic blood pressure in women were independent predictors of fibrin
d
-dimer levels. Since fibrin
d
-dimer does not appear to be independently related to many of the common cardiovascular risk factors, it may be a useful index of the thrombotic contribution to arterial disease.