bluuebunny/crossref_metadata_2025 huggingface dataset:
Segregation Analysis of Plasminogen Activator Inhibitor-1 and Fibrinogen Levels in the NHLBI Family Heart Study
Authors: Pankow James S. , Folsom Aaron R. , Province Michael A. , Rao D. C. , Williams Roger R. , Eckfeldt John , Sellers Thomas A.
Published: June 2011

Abstract—Elevated plasminogen activator inhibitor-1 (PAI-1) and fibrinogen concentrations are risk factors for coronary heart disease. We investigated environmental, familial, and genetic influences on PAI-1 antigen and fibrinogen concentrations in 2029 adults from 512 randomly ascertained families in 4 US communities. We used maximum-likelihood segregation analysis to fit several genetic and nongenetic modes of inheritance to the data to determine whether mendelian inheritance of a major gene could best explain the familial distributions of these 2 hemostatic factors. Age- and gender-adjusted familial correlations for PAI-1 antigen level averaged 0.16 in first-degree relatives (95% CI=0.11 to 0.21); the spouse correlation was positive but not statistically significant (r=0.10, 95% CI=−0.02 to 0.23). Complex segregation analysis indicated a major gene associated with higher PAI-1 concentrations in 65% of individuals from these families. Demographic, anthropometric, lifestyle, and metabolic characteristics together explained 37% to 47% of the variation in PAI-1 antigen levels, and the inferred major gene explained an additional 17% of the variance. Positive and statistically significant age- and gender-adjusted familial correlations in first-degree relatives indicated a possible heritable component influencing plasma fibrinogen concentration (r=0.17, 95% CI=0.13 to 0.22); however, segregation analysis did not provide statistical evidence of a major gene controlling fibrinogen level. These family data suggest that there are modest familial and genetic effects on the concentration of PAI-1.
Estradiol Suppresses MCP-1 Expression In Vivo
Authors: Pervin Shehla , Singh Rajan , Rosenfeld Michael E. , Navab Mohamad , Chaudhuri Gautam , Nathan Lauren
Published: June 2011

Abstract—The mechanisms by which 17β-estradiol retards atherogenesis are not known. The adhesion of monocytes to endothelial cells followed by the migration of monocytes into the artery wall are key cellular events that occur throughout the entire atherogenic process and may be responsive to estradiol. Monocyte chemoattractant protein-1 (MCP-1), a chemokine that is expressed in atherosclerotic lesions, is thought to play a major role in stimulating the migration of blood monocytes into developing atherosclerotic lesions. We therefore assessed the effects of estradiol in vivo on MCP-1 protein and mRNA expression in the descending thoracic aorta of rabbits fed a cholesterol-enriched (0.5%) diet for 6 weeks and in animals fed normal chow. MCP-1 protein was quantified by Western blot analysis and monocyte chemotaxis bioassay, and reverse transcription–polymerase chain reaction was used to ascertain the level of MCP-1 mRNA expression. We observed that in both ovary-intact and ovariectomized (OVX) animals, MCP-1 protein and mRNA expression were significantly increased by 6 weeks in animals fed a high-cholesterol diet. The cholesterol-induced increase in MCP-1 protein and mRNA expression was significantly attenuated in OVX rabbits supplemented with estradiol pellets (1.5- and 10.0-mg 60-day-release pellets), which yielded a range of estradiol concentrations encompassing the physiological levels. MCP-1 protein and mRNA expression were increased in normocholesterolemic OVX rabbits compared with normocholesterolemic ovary-intact animals, and this increase was prevented in OVX animals supplemented with estradiol pellets. Our observations indicate that both basal and hypercholesterolemia-induced increases in MCP-1 protein are modulated by physiological concentrations of estradiol.
Hypercholesterolemia Enhances Oxidant Production in Mesenteric Venules Exposed to Ischemia/Reperfusion
Authors: Kurose Iwao , Wolf Robert E. , Grisham Matthew B. , Granger D. Neil
Published: June 2011

Abstract
—It has been shown that hypercholesterolemia (HCh) exaggerates the microvascular dysfunction that is elicited by ischemia and reperfusion (I/R). The objective of this study was to determine whether oxidants contribute to the exaggerated inflammatory responses and enhanced albumin leakage observed in HCh rat mesenteric venules exposed to I/R (10 minutes of ischemia and 30 minutes of reperfusion). Intravital videomicroscopy was used to quantify the number of adherent and emigrated leukocytes, albumin extravasation, platelet-leukocyte aggregation in postcapillary venules, and the degranulation of adjacent mast cells. Oxidation of the fluorochrome dihydrorhodamine 123 (DHR) was used to monitor oxidant production by venular endothelium. I/R was shown to elicit an increased DHR oxidation in venules of both control and HCh rats, with the latter group exhibiting a significantly larger response. Treatment with either oxypurinol or superoxide dismutase largely prevented the leukocyte recruitment, platelet-leukocyte aggregation, mast cell degranulation, and enhanced DHR oxidation elicited by I/R in HCh rats. The enhanced albumin leakage was reduced by superoxide dismutase but not by oxypurinol. These results indicate that HCh amplifies the oxidant stress elicited by I/R and that interventions that blunt the oxidant stress effectively attenuate the leukocyte, platelet, and mast cell activation that result from I/R.
The Gln-Arg192 Polymorphism of Human Paraoxonase Gene Is Not Associated With Coronary Artery Disease in Italian Patients
Authors: Ombres Domenico , Pannitteri Gaetano , Montali Anna , Candeloro Antonio , Seccareccia Fulvia , Campagna Filomena , Cantini Renzo , Campa Pietro Paolo , Ricci Giorgio , Arca Marcello
Published: June 2011

Abstract
—Serum paraoxonase (PON) is an HDL-bound enzyme protecting LDL from oxidation. A common polymorphism of the paraoxonase gene (
PON1
) involving a Gln-to-Arg interchange at position 192 has been demonstrated to modulate PON activity toward paraoxon, a nonphysiological substrate; Arg192 (allele B) is associated with higher activity than Gln192 (allele A). This polymorphism has been proposed as a genetic marker of risk for coronary artery disease (CAD). However, the relationships between codon 192
PON1
genotypes, coronary atherosclerosis, and the occurrence of myocardial infarction (MI) are still controversial.
PON1
genotypes were determined in 472 consecutive subjects (>40 years old) who underwent coronary angiography. CAD (>50% stenosis) was detected in 310 subjects (CAD+); 162 subjects with <10% stenosis served as controls (CAD−). We also evaluated 204 randomly selected individuals as population controls.
PON1
genotypes were determined by PCR and
Alw
I restriction enzyme digestion. Frequencies of alleles A and B were 0.70 and 0.30 in angiographically assessed subjects and 0.73 and 0.27 in population controls, respectively (χ
2
=2.0;
P
<0.3). Distribution of
PON1
genotypes in CAD+ were not significantly different from those in CAD− (χ
2
=2.10;
P
<0.3). Similarly, no differences were observed in the subgroup of CAD+ with MI nor in that at higher oxidative risk (smokers and/or diabetics). After controlling for other coronary risk factors, no association was found between
PON1
alleles and the presence of CAD.
PON1
AA genotype was associated with reduced concentration of apolipoprotein B–containing triglyceride-rich lipoproteins. This study did not provide evidence of a significant association between codon 192
PON1
genotypes and coronary atherosclerosis in Italian patients. However, it did confirm that the
PON1
low-activity allele is associated with a less atherogenic lipid profile.
In Vitro–Differentiated Embryonic Stem Cell Macrophages
Authors: Moore Kathryn J. , Fabunmi Rosalind P. , Andersson Lorna P. , Freeman Mason W.
Published: June 2011

Abstract
—Monocytes/macrophages (Mø) appear to play a critical role in the initiation and progression of atherosclerotic lesions. In this study, we characterized in vitro–differentiated embryonic stem (ES) cell macrophages as a model system for studying atherosclerosis-associated Mø functions. Using immunofluorescence staining and Western analysis, we demonstrate that ES Mø express typical macrophage cell surface markers, as well as the known receptors for modified forms of low density lipoprotein (LDL), including the Mø scavenger receptors (SR-A type I and type II), CD36, and CD68. Differentiated ES Mø specifically bind and degrade
125
I-labeled acetylated LDL with high affinity, and their incubation with acetylated LDL (15 μg/mL) for 48 hours produces characteristic “foamy” Mø, as visualized by oil red O staining. ES Mø also express matrix-degrading metalloproteinases (MMP-3, MMP-9), which have been implicated in collagen breakdown in the fibrous cap of atherosclerotic plaques, and secrete cytokines (tumor necrosis factor-α, interleukin-6) in response to inflammatory stimuli. Transfection experiments, using a green fluorescent protein reporter gene, driven by the myeloid-specific promoter, CD11b, demonstrated that ES Mø can also be used to study macrophage-restricted gene expression in vitro. Taken together, these data demonstrate that ES Mø exhibit many properties typical of arterial lesion macrophages. Its ease of genetic manipulation makes it an attractive system for investigations of macrophage functions in vitro.
Tetraspanin CD9 Is Associated With Very Late–Acting Integrins in Human Vascular Smooth Muscle Cells and Modulates Collagen Matrix Reorganization
Authors: Scherberich Arnaud , Moog Sylvie , Haan-Archipoff Gisèle , Azorsa David O. , Lanza François , Beretz Alain
Published: June 2011

Abstract—CD9, a member of the tetraspanin family, and very late–acting (VLA) integrins are known to associate and form functional units on the surface of several cell types. We studied the changes in expression of CD9 and β1-integrins (CD29, VLA) in human vascular smooth muscle cells (VSMCs) under in vitro culture conditions mimicking proliferative vascular diseases. We also investigated possible interactions between CD9 and VLA integrins in VSMCs. We found that CD9 is highly expressed in VSMCs and is subject to modulation, depending on the proliferative/contractile state of the cells. In the contractile phenotype, the levels of CD9, CD81, another tetraspanin, and CD29 are ≈50% of those found in the proliferative phenotype. Coimmunoprecipitation experiments showed physical association between CD9 and CD29. CD9 was mainly associated with α2and α3-integrins (CD49b and c) and also with α5-integrin to a weaker extent. Functionally, the addition of anti-CD9 monoclonal antibodies (MoAbs) doubled the extent of collagen gel contraction mediated by VSMCs, a model for the reorganization of the extracellular collagen matrix occurring in the vessel wall. Anti-CD29 MoAbs inhibited gel contraction, but anti-CD9 MoAbs counteracted this inhibitory effect of anti-CD29 MoAbs. Transfection of human CD9 into Chinese hamster ovary cells more than doubled the extent of Chinese hamster ovary cell–mediated collagen gel contraction (130% stimulation), confirming a role for CD9 in extracellular matrix reorganization. Thus, CD9 seems to be involved in the modulation of VLA integrin–mediated collagen matrix reorganization by VSMCs. These findings suggest that high CD9 expression is associated with a proliferative state of VSMCs. The role of CD9 could be to modulate the function of VLA integrins on the surface of VSMCs.
Effects of β3-Integrin Blockade (c7E3) on the Response to Angioplasty and Intra-Arterial Stenting in Atherosclerotic Nonhuman Primates
Authors: Deitch Jonathan S. , Williams J. Koudy , Adams Michael R. , Fly Christopher A. , Herrington David M. , Jordan Robert E. , Nakada Marian T. , Jakubowski Joseph A. , Geary Randolph L.
Published: June 2011

Abstract
—Because the β3-antagonist abciximab (c7E3 Fab) has significantly improved late outcomes after coronary angioplasty, the β3 integrins have been implicated in the arterial response to injury. However, the mechanisms underlying this benefit are unknown. The observation that c7E3 binds β3 integrins on vascular cells (αvβ3) with affinity equal to that for the platelet glycoprotein IIb/IIIa integrin has led to the hypothesis that c7E3 may act directly on the artery wall to prevent restenosis after angioplasty. To test this hypothesis, we studied the effects of c7E3 on structural changes within the artery wall after angioplasty or stent angioplasty in 23 male cynomolgus monkeys with established atherosclerosis. Animals were randomly assigned to receive either a bolus of c7E3 (0.4 mg/kg IV, n=11) followed by a 48-hour infusion (0.2 μg · kg
−1
· min
−1
) or an equal volume of vehicle (n=12). Animals received weight-adjusted aspirin and heparin and then underwent unilateral iliac artery experimental angioplasty and subclavian artery stent angioplasty (Palmaz). Iliac artery lumen diameter (LD) was determined by angiography at baseline (LD
Pre
), after angioplasty (LD
Post
), and 35 days later (LD
Day35
). Arteries were then fixed by perfusion and removed for analysis. Lumen, intima, media, and external elastic lamina (EEL) areas were measured in iliac artery cross sections. Values from each injured iliac artery were normalized to the contralateral uninjured iliac artery to control for interanimal variability in baseline artery size and atherosclerosis extent. Intimal area was also measured in subclavian stent cross sections. c7E3 blocked platelet aggregation and prolonged the bleeding time from 2.8±1.1 to 19.8±2.5 minutes,
P
<0.001. Experimental angioplasty increased LD
Post
an average of 28%, and the initial gain was similar in both groups (
P
=NS). Despite an anti-platelet effect, c7E3 did not inhibit iliac lumen narrowing (LD
Day35
−LD
Post
: c7E3, −0.69±0.17 versus vehicle, −0.99±.17 mm,
P
=0.35); intimal hyperplasia (neointima area: c7E3, 1.12±.28 versus vehicle, 1.22±.20 mm
2
,
P
=0.77); or decrease in artery wall size (EEL area [percent of uninjured control]: c7E3, 101±7% versus vehicle, 121±7%). Stent intimal hyperplasia was also unaltered by c7E3 treatment (neointimal area: c7E3, 1.09±0.16 versus vehicle, 1.28±0.11 mm
2
,
P
=0.36). These results suggest that the benefits of c7E3 treatment in coronary angioplasty were not from inhibition of intimal hyperplasia or improved artery wall remodeling. Alternative mechanisms should be explored to explain improved late outcomes after angioplasty in patients treated with c7E3.
Sequences Within the Amino Terminus of ApoB100 Mediate Its Noncovalent Association With Apo(a)
Authors: Gabel Brent R. , McLeod Roger S. , Yao Zemin , Koschinsky Marlys L.
Published: June 2011

Abstract—Although sequences within theCterminus of apolipoprotein B (apoB) have been implicated in the formation of covalent lipoprotein(a) [Lp(a)] particles, sequences in apoB that mediate initial noncovalent interaction with apo(a) remain to be characterized. To address this question, we have used an affinity chromatography method in which 2 recombinant forms of apo(a) [r-apo(a); either a 17-kringle form (17K) or a derivative containing apo(a) kringle IV types 5-8] have been immobilized onto Sepharose beads. Conditioned media from rat hepatoma (McA-RH7777) cell lines stably expressing various carboxyl-terminally truncated forms of human apoB (ranging from full-length apoB to apoB15) were applied to the r-apo(a) affinity columns; the columns were subsequently washed and eluted with ε-aminocaproic acid (ε-ACA). Specific binding was quantified by Western blot analysis of column fractions. Of the apoB truncations examined, apoB94, apoB42, apoB37, and apoB29 exhibited complete specific binding to 17K r-apo(a). Only ≈50% binding was observed for apoB18, whereas essentially no detectable binding was observed with apoB15. In all cases, similar results were obtained when the r-apo(a) kringle IV types 5-8–Sepharose column was used. Additionally, substitution of proline for ε-ACA as the eluent resulted in similar column profiles with either r-apo(a) affinity column. We also demonstrated that apoB48 present in chylomicrons bound completely to the 17K column in an ε-ACA–dependent manner. Taken together, these results represent the first demonstration thatN-terminal sequences in apoB between amino acid residues 680 (apoB15) and 781 (apoB18) are essential for noncovalent association with apo(a) and that these sequences interact with domain(s) present within apo(a) kringle IV types 5-8.
Opposite Effects of Plasma Homocysteine and the Methylenetetrahydrofolate Reductase C677T Mutation on Carotid Artery Geometry in Asymptomatic Adults
Authors: Demuth Karine , Moatti Nicole , Hanon Olivier , Benoit Marie Odile , Safar Michel , Girerd Xavier
Published: June 2011

Abstract
—Studies of symptomatic patients have identified hyperhomocysteinemia as an independent risk factor for vascular disease. In case-control studies, a point mutation (C677T) in the gene encoding 5,10-methylenetetrahydrofolate reductase (MTHFR) has also been linked to an increased risk of vascular disease through its effect on homocysteinemia. Our aim was to extend these observations to asymptomatic subjects by studying the influence of both homocysteinemia and its mutation on carotid artery geometry. We examined 144 subjects free of atherosclerotic lesions. Fasting homocysteinemia was measured by high-performance liquid chromatography with fluorometric detection. MTHFR genotype was analyzed by polymerase chain reaction followed by
Hin
fI digestion. Carotid artery geometry was characterized by internal diameter and intima-media thickness, as assessed by a high-resolution echo-tracking system. Subjects in the upper homocysteine tertile had a greater carotid internal diameter than did subjects in the middle and lower tertiles (6516±770 versus 6206±641 and 5985±558 μm, respectively;
P
<0.001). Subjects homozygous for the mutation had a smaller carotid artery internal diameter than did subjects heterozygous or homozygous for the wild-type allele (5846±785 versus 6345±673 and 6199±671 μm, respectively;
P
<0.05). Homocysteinemia was not significantly increased in subjects homozygous for the mutation. In multivariate regression analysis, homocysteinemia was independently and positively associated with lumen diameter (
P
=0.0008) and wall thickness (
P
=0.020). Conversely, homozygosity for the mutation was negatively associated with internal diameter (
P
=0.009). These preliminary data suggest that mildly elevated homocysteinemia and homozygosity for the MTHFR C677T mutation are associated with opposite preclinical modifications of carotid artery geometry. If confirmed, these results may have important implications for new treatment strategies for vascular disease before the onset of clinical manifestations.
The HMG-CoA Reductase Inhibitor Atorvastatin Increases the Fractional Clearance Rate of Postprandial Triglyceride-Rich Lipoproteins in Miniature Pigs
Authors: Burnett John R. , Barrett P. Hugh R. , Vicini Paolo , Miller David B. , Telford Dawn E. , Kleinstiver Sandra J. , Huff Murray W.
Published: June 2011

Abstract
—We have previously shown in vivo that the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin decreases hepatic apolipoprotein B (apoB) secretion into plasma. To test the hypothesis that atorvastatin modulates exogenous triglyceride-rich lipoprotein (TRL) metabolism in vivo, an oral fat load (2 g fat/kg body wt) containing retinol (50 000 IU) was given to 6 control miniature pigs and to 6 animals after 28 days of treatment with atorvastatin 3 mg · kg
−1
· d
−1
. A multicompartmental model was developed by use of SAAM II and kinetic analysis performed on the plasma retinyl palmitate (RP) data. Peak TRL (
d
<1.006 g/mL; S
f
>20) triglyceride concentrations were decreased 29% by atorvastatin, and the time to achieve this peak was delayed (5.2 versus 2.3 hours;
P
<0.01). The TRL triglyceride 0- to 12-hour area under the curve was decreased by 24%. In contrast, atorvastatin treatment had no effect on peak TRL RP concentrations, time to peak, or its rate of appearance into plasma; however, the TRL RP 0- to 12-hour area under the curve was decreased by 20%. Analysis of the RP kinetic parameters revealed that the TRL fractional clearance rate was increased significantly, 1.4-fold (3.093 versus 2.276 pools/h;
P
=0.012), with atorvastatin treatment. The percent conversion of TRL RP from the rapid-turnover to the slow-turnover compartment was decreased by 47% with atorvastatin treatment. The TRL RP fractional clearance rate was negatively correlated with very low density lipoprotein apoB production rate measured in the fasting state (
r
=−0.49). Thus, although atorvastatin had no effect on intestinal TRL assembly and secretion, plasma TRL clearance was significantly increased, an effect that may relate to a decreased competition for removal processes by hepatic very low density lipoprotein.
Uptake of Oxidized LDL by Macrophages Results in Partial Lysosomal Enzyme Inactivation and Relocation
Authors: Li Wei , Yuan Xi Ming , Olsson Anders G. , Brunk Ulf T.
Published: June 2011

Abstract—The cytotoxicity of oxidized LDL (oxLDL) to several types of artery wall cells might contribute to atherosclerosis by causing cell death, presumably by both apoptosis and necrosis. After its uptake into macrophage lysosomes by receptor-mediated endocytosis, oxLDL is poorly degraded, resulting in ceroid-containing foam cells. We studied the influence of oxLDL on lysosomal enzyme activity and, in particular, on lysosomal membrane stability and the modulation of these cellular characteristics by HDL and vitamin E (vit-E). Unexposed cells and cells exposed to acetylated LDL (AcLDL) were used as controls. The lysosomal marker enzymes cathepsin L andN-acetyl-β-glucosaminidase (NAβGase) were biochemically assayed in J-774 cells after fractionation. Lysosomal integrity in living cells was assayed by the acridine orange (AO) relocation test. Cathepsin D was immunocytochemically demonstrated in J-774 cells and human monocyte-derived macrophages. We found that the total activities of NAβGase and cathepsin L were significantly decreased, whereas their relative cytosolic activities were enhanced, after oxLDL exposure. Labilization of the lysosomal membranes was further proven by decreased lysosomal AO uptake and relocation to the cytosol of cathepsin D, as estimated by light and electron microscopic immunocytochemistry. HDL and vit-E diminished the cytotoxicity of oxLDL by decreasing the lysosomal damage. The results indicate that endocytosed oxLDL not only partially inactivates lysosomal enzymes but also destabilizes the acidic vacuolar compartment, causing relocation of lysosomal enzymes to the cytosol. Exposure to AcLDL resulted in its uptake with enlargement of the lysosomal apparatus, but the stability of the lysosomal membranes was not changed.
Altered Myocardial Vasodilatation in Patients With Hypertriglyceridemia in Anatomically Normal Coronary Arteries
Authors: Yokoyama Ikuo , Ohtake Tohru , Momomura Shin-ichi , Yonekura Katsunori , Kobayakawa Naoshi , Aoyagi Teruhiko , Sugiura Seiryo , Sasaki Yasuhito , Omata Masao
Published: June 2011

Abstract
—Reduced myocardial vasodilatation (MVD) in hypercholesterolemics without overt coronary stenosis has been reported. However, the status of MVD in hypertriglyceridemics has not yet been clarified. The aim of this study was to investigate whether MVD is impaired in patients with hypertriglyceridemia without overt coronary stenosis. Twenty-three hypertriglyceridemics (10 normocholesterolemic hypertriglyceridemics [HTGs] and 13 mixed combined hyperlipidemics [MCHLs]) and 13 age-matched controls were studied. All patients were proven to have more than one normal coronary artery, as diagnosed by coronary angiography, and those segments that were perfused by anatomically normal coronary arteries were used in the study. Myocardial blood flow (MBF) during dipyridamole (DP) loading and baseline MBF were measured by using positron emission tomography and [
13
N]ammonia, after which MVD was calculated. Baseline MBF (mL · min
−1
· 100 g
−1
) was comparable among HTG (76.0±26.1), MCHL (77.0±26.1), and controls (80.3±38.5). However, MBF during DP loading was significantly lower in MCHL (159±52.5) than in control subjects (292±166,
P
<.01), while it was comparable in HTG (202±104) and controls. MVD was significantly reduced in both HTG (2.70±1.09,
P
<.05) and MCHL (2.07±.70,
P
<.01) compared with controls (3.73±1.14). MVD in MCHLs tended to be reduced compared with that in HTGs, but the difference was statistically insignificant (
P
=.08). There was a significant relationship between MVD and both plasma triglycerides (
r
=−.47,
P
<.01) and plasma total cholesterol (
r
=−.55,
P
<.01). When controls and HTGs were combined, the relationship between MVD and plasma total triglycerides became more prominent (
r
=−.55,
P
<.05), and the significant relationship between cholesterol level and MVD disappeared. Multivariate regression analysis has revealed that the triglyceride level (F=5.2,
P
<.05) was independently related to MVD (
r
=.69,
P
<.01). In conclusion, MVD was reduced in hypertriglyceridemics in anatomically normal coronary arteries. Hypertriglyceridemia is an independent factor for this abnormality.
Calcification of Human Vascular Cells In Vitro Is Correlated With High Levels of Matrix Gla Protein and Low Levels of Osteopontin Expression
Authors: Proudfoot Diane , Skepper Jeremy N. , Shanahan Catherine M. , Weissberg Peter L.
Published: June 2011

Abstract—The cellular and molecular events leading to calcification in atherosclerotic lesions are unknown. We and others have shown that bone-associated proteins, particularly matrix Gla protein (MGP) and osteopontin (OP), can be detected in atherosclerotic lesions, thus suggesting an active calcification process. In the present study, we aimed to determine whether human vascular smooth muscle cells (VSMCs) could calcify in vitro and to determine whether MGP and OP have a role in vascular calcification. We established that human aortic VSMCs and placental microvascular pericytes spontaneously form nodules in cell culture and induce calcification, as detected by von Kossa’s method, Alizarin red S staining, and electron microscopy. The cells in calcifying nodules differed from those in monolayer cultures by expressing higher levels of the SMC markers α-SM actin, SM22α, and calponin. In addition, Northern blot analysis revealed that in human VSMCs, calcification was associated with increased levels of MGP mRNA. In contrast, OP mRNA was barely detectable in calcified human VSMCs and pericyte nodules, nor was OP protein detected, suggesting that OP was not necessary for calcification to occur. These studies reveal that human VSMCs are capable of inducing calcification and that MGP may have a role in human vascular calcification.
A Novel Homozygous Missense Mutation in the Apo A-I Gene With Apo A-I Deficiency
Authors: Huang Wei , Sasaki Jun , Matsunaga Akira , Nanimatsu Hiroshi , Moriyama Kengo , Han Hua , Kugi Mari , Koga Takafumi , Yamaguchi Kohei , Arakawa Kikuo
Published: June 2011

Abstract
—We analyzed the genetic defect in a 67-year-old Japanese male patient with apolipoprotein (apo) A-I and high density lipoprotein (HDL) deficiencies, corneal opacities, and coronary artery disease. The plasma concentrations of apoA-I and HDL cholesterol were 2.9 to 7.3 mg/dL and 0.08 to 0.19 mmol/L, respectively. The lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol esterification rate were <40% of normal control values. LCAT mass was ≈50% of normal control. Sequence analysis of polymerase chain reaction–amplified DNA of the proband’s apoA-I gene showed a homozygous T-to-A transition resulting in the substitution of Val 156 with Glu (apoA-I Oita). Direct sequencing of samples obtained from other family members showed that the brother was homozygous, whereas the son was a heterozygous carrier of apoA-I Oita. The heterozygote for apo A-I Oita showed nearly 60% of normal apoA-I and normal HDL cholesterol levels. In vivo turnover studies in rabbits demonstrated that the variant apoA-I was rapidly cleared from plasma compared with normal human apoA-I. Our data suggest that the Val156Glu substitution is associated with apoA-I and HDL deficiency, partial LCAT deficiency, and corneal opacities and that Val156 of apoA-I may play an important role in apoA-I function.
Plasma Lipoproteins Support Prothrombinase and Other Procoagulant Enzymatic Complexes
Authors: Moyer Martin P. , Tracy Russell P. , Tracy Paula B. , Veer Cornelis van’t , Sparks Charles E. , Mann Kenneth G.
Published: June 2011

Abstract—The prothrombinase complex (factor [F]Xa, FVa, calcium ions, and lipid membrane) converts prothrombin to thrombin (FIIa). To determine whether plasma lipoproteins could provide a physiologically relevant surface, we determined the rates of FIIa production by using purified human coagulation factors, and isolated fasting plasma lipoproteins from healthy donors. In the presence of 5 nmol/L FVa, 5 nmol/L FXa, and 1.4 μmol/L prothrombin, physiological levels of very low density lipoprotein (VLDL) (0.45 to 0.9 mmol/L triglyceride, or 100 to 200 μmol/L phospholipid) yielded rates of 2 to 8 nmol FIIa · L−1· s−1in a donor-dependent manner. Low density lipoprotein (LDL) and high density lipoprotein (HDL) also supported prothrombinase but at much lower rates (≤1.0 nmol FIIa · L−1· s−1). For comparison, VLDL at 2 mmol/L triglyceride yielded ≈50% the activity of 2×108thrombin-activated platelets per milliliter. Although the FIIa production rate was slower on VLDL than on synthetic phosphatidylcholine/phosphatidylserine vesicles (≈50 nmol FIIa · L−1· s−1), the prothrombinKmvalues were similar, 0.8 and 0.5 μmol/L, respectively. Extracted VLDL lipids supported rates approaching those of phosphatidylcholine/phosphatidylserine vesicles, indicating the importance of the intact VLDL conformation. However, the presence of VLDL-associated, factor-specific inhibitors was ruled out by titration experiments, suggesting a key role for lipid organization. VLDL also supported FIIa generation in an assay system comprising 0.1 nmol/L FVIIa; 0.55 nmol/L tissue factor; physiological levels of FV, FVIII, FIX, and FX; and prothrombin (3 nmol/L FIIa · L−1· s−1). These results indicate that isolated human VLDL can support all the components of the extrinsic coagulation pathway, yielding physiologically relevant rates of thrombin generation in a donor-dependent manner. This support is dependent on the intact lipoprotein structure and does not appear to be regulated by specific VLDL-associated inhibitors. Further studies are needed to determine the extent of this activity in vivo.
LDL Induces Transcription Factor Activator Protein-1 in Human Endothelial Cells
Authors: Zhu Yi , Lin Jane H.-C. , Liao Hai-Ling , Friedli Otto , Verna Lynne , Marten Norman W. , Straus Daniel S. , Stemerman Michael B.
Published: June 2011

Abstract—Low density lipoprotein (LDL) has been shown to perturb endothelial cells, with manifestations ranging from alterations in free radicals and arachidonate metabolism to stress fiber formation and monocyte recruitment. Some of these changes are regulated by LDL at the transcriptional level. Using mobility shift assays with consensus sequences for various transcription factors, we have detected an increase in activator protein 1 (AP-1), but not nuclear factor-κB (NF-κB), binding in human umbilical vein endothelial cells exposed to LDL. Following transfection, AP-1–driven chloramphenicol acetyltransferase and AP-1–driven-luciferase are upregulated by LDL. In contrast, there is no effect on NF-κB–driven chloramphenicol acetyltransferase. AP-1 increases in a biphasic fashion, with the first peak occurring 6 hours after and the second 48 hours after exposure to LDL. This AP-1 binding increase involves c-Jun, but not c-Fos, as shown by gel supershift, Northern hybridization, and Western blotting analyses. c-Jun mRNA levels are elevated by 9 hours after and remain so until at least 24 hours after exposure to LDL. c-Jun protein levels increase at 12 hours and continue to rise for 24 hours after exposure to LDL. Moreover, this LDL-increased AP-1 binding is suppressed by several protein kinase (PK) inhibitors: the PKC inhibitor calphostin C, the cAMP-dependent PK inhibitor H89, and the tyrosine PK inhibitors genistein and lavendustin A. This study demonstrates that (1) LDL is an endothelial agonist distinct from other cell stimulators, such as cytokines, endotoxin, and phorbol 12-myristate 13-acetate, because LDL appears to activate human umbilical vein endothelial cells predominantly through the transcription factor AP-1 and not NF-κB; and (2) LDL increases AP-1 via mechanisms involving multiple kinase activities and c-Jun transcription.
Influence of Biochemical Alterations on Arterial Stiffness in Patients With End-stage Renal Disease
Authors: Blacher Jacques , Demuth Karine , Guerin Alain P. , Safar Michel E. , Moatti Nicole , London Gérard M.
Published: June 2011

Abstract
—The incremental elastic modulus of the common carotid and radial arteries is increased in patients with end-stage renal disease (ESRD), independently of blood pressure, wall stress, and the presence of atherosclerotic alterations. Whether biochemical factors may be involved in the arterial changes and related to renal dysfunction remain largely ignored. To assess this question, we measured aortic (carotid-femoral), upper-limb (carotid-radial), and lower-limb (femoral-tibial) pulse wave velocity (PWV) in 74 ESRD patients undergoing hemodialysis in comparison with 57 control subjects similar in age, sex ratio, and mean blood pressure. We evaluated arterial blood presure by sphygmomanometry, aortic calcifications and cardiac mass by echography, and routine biochemical parameters, total plasma homocysteine, and plasma endothelin levels by standard techniques. In the population of patients with ESRD, on the basis of multiple stepwise regression analysis, aortic PWV was positively and independently correlated with systolic blood pressure (
P
<.0001), age (
P
<.0001), prevalence of aortic calcification (
P
=.0004), and the prevalence of diabetes mellitus (
P
=.0043). Upper-limb PWV was influenced exclusively by mean blood pressure (
P
<.0001). Lower-limb PWV was positively and independently correlated with plasma total homocysteine (
P
=.0004) and plasma endothelin (
P
=.0187) only. At any vascular site, PWV was not independently correlated with tobacco consumption; plasma levels of cholesterol, triglyceride, fibrinogen, or hemoglobin; body mass index; or the presence of bilateral nephrectomy. Finally, plasma homocysteine was independently correlated with cardiac mass (
P
=.0022). This study provides evidence that in ESRD patients, the stiffness of the arterial wall and cardiac mass are strongly influenced by biochemical factors related to the kidney alterations and are independent of age and blood pressure level. Increased plasma endothelin and homocysteine may be specifically involved in the vascular damage of lower limbs.
Biphasic Regulation of Transcription Factor Nuclear Factor-κB Activity in Human Endothelial Cells by Lysophosphatidylcholine Through Protein Kinase C–Mediated Pathway
Authors: Sugiyama Seigo , Kugiyama Kiyotaka , Ogata Nobuhiko , Doi Hideki , Ota Yasutaka , Ohgushi Masamichi , Matsumura Toshiyuki , Oka Hideki , Yasue Hirofumi
Published: June 2011

Abstract—Lysophosphatidylcholine (lysoPC), which is generated in oxidized LDL (Ox-LDL) and abundantly exists in atherosclerotic arterial walls, has been shown to alter various endothelial functions and induces several endothelial genes expressed in atherosclerotic arterial walls. Nuclear factor-kappa B (NF-κB), a pleiotropic transcription factor, plays an important role in regulation of expression of various genes implicated in atherosclerosis. We have previously reported that lysoPC transferred from Ox-LDL to endothelial surface membrane activates endothelial protein kinase C (PKC), leading to modulated endothelial functions. This study was aimed at determining whether lysoPC could modulate activity of transcription factors in cultured human umbilical vein endothelial cells (HUVECs) by using electrophoretic mobility shift assay. LysoPC was found to increase DNA-binding activity of NF-κB in HUVECs within 15 minutes, which peaked at 1 to 2 hours and subsequently declined to the baseline level at 6 hours. Lower concentrations (5 to 15 μmol/L) of lysoPC markedly increased NF-κB activity, but higher concentration (50 μmol/L) of lysoPC inhibited the activity. Phorbol 12-myristate 13-acetate, a potent activator of PKC, also augmented NF-κB activity in HUVECs, mimicking the effects of lysoPC; furthermore, calphostin C and chelerythrine chloride, specific PKC inhibitors, and α-tocopherol, a clinically potent PKC inhibitor, suppressed the lysoPC-induced NF-κB activation. These results indicate that lysoPC regulates NF-κB activity in a biphasic manner dependent on its concentrations and incubation time in human endothelial cells and the endothelial PKC activation may in part be involved in the lysoPC-induced NF-κB activation. Thus, the time course and the positive and negative biphasic regulatory actions of lysoPC on NF-κB activity in endothelial cells might exhibit a unique effect of lysoPC in arterial walls on the different stages of atherosclerosis.
Molecular Genetic Study of Finns With Hypoalphalipoproteinemia and Hyperalphalipoproteinemia
Authors: Miettinen H.E. , Gylling H. , Tenhunen J. , Virtamo J. , Jauhiainen M. , Huttunen J.K. , Kantola I. , Miettinen T.A. , Kontula K.
Published: June 2011

Abstract—In an attempt to identify genetic factors underlying extreme alterations of serum HDL cholesterol (HDL-C) concentrations, we examined two probands with HDL-C levels <0.2 mmol/L and subsequently screened two large cohorts of smoking men, one with very low (0.2 to 0.7 mmol/L, n=156) and the other with elevated (1.9 to 3.6 mmol/L, n=160) HDL-C levels, for the newly detected mutations as well as some other mutations proposed to affect HDL-C levels. One of the probands had corneal opacities, microalbuminuria, hypertriglyceridemia, and reduced LDL apoprotein B concentration; the other had anemia and presented with stomatocytosis in his peripheral blood. The first proband was found to be homozygous for a novel LCAT Gly230Arg (LCATFin) mutation, and the second was homozygous for an Arg399Cys mutation we described previously. Transient expression of the mutant LCATFincDNA in COS cells disclosed markedly diminished LCAT enzyme activity. In the low–HDL-C group of men (n=156), 8 carriers of LCATFinand 1 carrier of the LCAT Arg399Cys were identified. In addition, the frequency of the lipoprotein lipase (LPL) Asn291Ser mutation was significantly (P<.05) higher in the low–HDL-C group (4.8%) than in the high–HDL-C group (1.6%). In addition, we identified 1 carrier of the intron 14G→A mutation of cholesterol ester transfer protein (CETP) in the high–HDL-C group and subsequently demonstrated cosegregation of the mutant allele with elevated HDL-C levels in the proband’s family. In conclusion, we have identified a novel LCAT gene Gly230Arg mutation (LCATFin), which, together with the LPL Asn291Ser mutation, represents a relatively common genetic cause of diminishing HDL-C levels, at least among Finns. This article also reports occurrence of a CETP mutation in subjects having non-Japanese roots.
Elevated Plasma Fibrinogen
Authors: van der Bom Johanna G. , de Maat Moniek P. M. , Bots Michiel L. , Haverkate Frits , de Jong P. T. V. M. , Hofman Albert , Kluft Cornelis , Grobbee Diederick E.
Published: June 2011

Abstract
—An association between increased plasma fibrinogen and an increased risk for myocardial infarction (MI) is well established, but the nature of this association is subject to debate. Our aim was to shed light on the potentially causal nature of this association. We examined whether increased plasma fibrinogen, due to a condition that is independent of cardiovascular events, also increases the risk for MI. A case-control study was performed in 139 subjects with a history of MI and 287 control subjects selected from the Rotterdam Study, a population-based cohort of 7983 subjects aged 55 years and older. The genotype of the −455G/A polymorphism in the fibrinogen β-gene was determined by polymerase chain reaction. Functional plasma fibrinogen levels were determined according to von Clauss. The plasma level of fibrinogen was significantly higher in subjects with one or two
A
alleles compared with subjects with the
GG
genotype: 3.8 (95% confidence interval [CI], 3.6 to 3.9) g/L and 3.6 (3.5 to 3.7) g/L, respectively. With increasing plasma fibrinogen level, the risk for MI increased gradually; a rise in fibrinogen of 1 g/L was associated with a 45% increased risk (odds ratio adjusted for age, sex, and smoking, 1.45; 95% CI, 1.12 to 1.88). There was no association between the genotype of the −455G/A polymorphism and the risk for MI. The −455G/A polymorphism is therefore associated with increased plasma fibrinogen levels but not with an increased risk for MI. These findings indicate that an increased plasma fibrinogen level due to this genetic factor does not increase the risk for MI.
Increased Degradation of Lipoprotein(a) in Atherosclerotic Compared With Nonlesioned Aortic Intima–Inner Media of Rabbits
Authors: Nielsen Lars B. , Juul Klaus , Nordestgaard Børge G.
Published: June 2011

Abstract
—To investigate a potential role of lipoprotein(a) [Lp(a)] in foam cell formation, we have measured the degradation rates of Lp(a) and LDL in the rabbit aorta in vivo. Lp(a) (or LDL) was labeled with both
131
I-TC and
125
I and injected into 17 rabbits with extensive aortic atherosclerosis and into 16 rabbits without atherosclerosis. As the protein moiety of the doubly labeled lipoproteins is degraded,
131
I-TC is trapped in the cell, whereas
125
I diffuses out of the cell. Twenty-four hours after injection, 12 samples of the aorta and biopsies from 9 other tissues were removed. The degradation rate of Lp(a) (percent of plasma pool per gram tissue per day) was less than that of LDL in the adrenals and in the intestine. In contrast, degradation rates of Lp(a) and LDL were similar in liver, spleen, kidney, heart, lung, skeletal muscle, and adipose tissue. In nonlesioned aortic intima–inner media, the degradation rate of Lp(a) was 39% of that of LDL (
t
test:
P
<.05 in aortic arch and thoracic aorta), whereas the degradation rates of Lp(a) and LDL were similar in atherosclerotic aortic intima–inner media. Lp(a) degradation rates were markedly increased in atherosclerotic compared with nonlesioned aortic intima–inner media: 28.2±9.2×10
−7
% and 5.0±0.6×10
−7
% of the plasma pool per gram tissue per day in the intima–inner media of the proximal segment of atherosclerotic and nonlesioned aorta, respectively (
t
test:
P
<.01). These results suggest that the metabolism of Lp(a) is different from that of LDL in nonlesioned arterial intima, possibly reflecting that Lp(a) is degraded by LDL receptors in arterial intima less efficiently than LDL. The results also indicate that the degradation rate of Lp(a) is markedly increased in atherosclerotic lesions of rabbits, supporting the idea that Lp(a) contributes to foam cell formation during the development of atherosclerosis.
Binding of Human Urokinase-Type Plasminogen Activator to Its Receptor
Authors: Quax Paul H. A. , Grimbergen Jos M. , Lansink Mirian , Bakker Arjen H. F. , Blatter Marie-Claude , Belin Dominique , van Hinsbergh Victor W. M. , Verheijen Jan H.
Published: June 2011

Abstract
—Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated
125
I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate—treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated
125
I-labeled mutant UPA could not bind to phorbol myristate acetate–treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated
125
I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no effect on binding to UPAR on human ECs. The functional integrity of the growth factor domain in the non–receptor binding Asn22Tyr mutant is suggested by the fact that binding of this mutant to a murine UPAR can be restored after additional mutations in the growth factor domain, Asn27,His29,Trp30 to Arg27,Arg29,Arg30. We conclude that Asn22 and Asn27,His29,Trp30 in human UPA are key determinants in the species-specific binding of the enzyme to its receptor and that changing Asn22 into Tyr results in a UPA mutant with strongly reduced binding to UPAR.
Effect of Thyroid Function on LDL Oxidation
Authors: Costantini Fabrizio , Pierdomenico Sante D. , Cesare Domenico De , De Remigis Pierluigi , Bucciarelli Tonino , Bittolo-Bon Gabriele , Cazzolato Giuseppe , Nubile Giuseppe , Guagnano Maria T. , Sensi Sergio , Cuccurullo Franco , Mezzetti Andrea
Published: June 2011

Abstract
—In this study, the effect of different levels of thyroid hormone and metabolic activity on low density lipoprotein (LDL) oxidation was investigated. Thus, in 16 patients with hyperthyroidism, 16 with hypothyroidism, and 16 age- and sex-matched healthy normolipidemic control subjects, the native LDL content in lipid peroxides, vitamin E, β-carotene, and lycopene, as well as the susceptibility of these particles to undergo lipid peroxidation, was assessed. Hyperthyroidism was associated with significantly higher lipid peroxidation, as characterized by a higher native LDL content in lipid peroxides, a lower lag phase, and a higher oxidation rate than in the other two groups. This elevated lipid peroxidation was associated with a lower LDL antioxidant concentration. Interestingly, hypothyroid patients showed an intermediate behavior. In fact, in hypothyroidism, LDL oxidation was significantly lower than in hyperthyroidism but higher than in the control group. Hypothyroidism was also characterized by the highest β-carotene LDL content, whereas vitamin E was significantly lower than in control subjects. In hyperthyroidism but not in the other two groups, LDL oxidation was strongly influenced by free thyroxine blood content. In fact in this group, the native LDL lipid peroxide content and the lag phase were directly and indirectly, respectively, related to free thyroxine blood levels. On the contrary, in hypothyroidism LDL oxidation was strongly and significantly related to serum lipids. In conclusion, both hypothyroidism and hyperthyroidism are characterized by higher levels of LDL oxidation when compared with normolipidemic control subjects. In hyperthyroid patients, the increased lipid peroxidation was strictly related to free thyroxine levels, whereas in hypothyroidism it was strongly influenced by serum lipids.