bluuebunny/crossref_metadata_2025 huggingface dataset:
Genotype-Specific Transcriptional Regulation of PAI-1 Expression by Hypertriglyceridemic VLDL and Lp(a) in Cultured Human Endothelial Cells
Authors: Li Xin-Nong , Grenett Hernan E. , Benza Raymond L. , Demissie Sossiena , Brown Stephen L. , Tabengwa Edlue M. , Gianturco Sandra H. , Bradley William A. , Fless Gunther M. , Booyse Francois M.
Published: June 2011

Abstract
The hypothesized relationships between plasminogen activator inhibitor (PAI-1) genotypes, PAI-1 levels, and their potential regulation by hypertriglyceridemic (HTG) very low density lipoprotein (VLDL) and lipoprotein(a) [Lp(a)] was examined in a PAI-1 genotyped human umbilical vein endothelial cell (HUVEC) culture model system. Individual human umbilical veins were used to obtain cultured ECs and were genotyped for PAI-1 by using the
Hin
dIII restriction fragment length polymorphism (RFLP) as a marker for genetic variation. Digested genomic DNA, examined by Southern blot analysis and probed with an [α-
32
P]dCTP–labeled 2.2-kb PAI-1 cDNA, yielded three RFLPs designated 1/1 (22-kb band only), 1/2 (22- plus 18-kb bands), and 2/2 (18-kb band only). Individual PAI-1 genotyped HUVEC cultures were incubated in the absence or presence of HTG-VLDL (0 to 50 μg/mL) or Lp(a) (0 to 50 μg/mL) at 37°C for various times (4 to 24 hours), followed by analyses of PAI-1 antigen (by ELISA) and mRNA (by ribonuclease protection assay) levels, EC surface–localized plasmin generation assays, and nuclear run-on transcription assays. Secreted PAI-1 antigen levels were increased ≈2- to 3-fold by HTG-VLDL and ≈1.6 to 2-fold by Lp(a); mRNA levels were increased ≈3- to 4.5-fold by HTG-VLDL and ≈2.5- to 3.2-fold by Lp(a) compared with medium-incubated controls, primarily in the 2/2 PAI-1 genotype HUVEC cultures. Increases in PAI-1 mRNA induced by HTG-VLDL or Lp(a) could be abolished by coincubation with actinomycin D (2×10
−6
mol/mL) or puromycin (1 μg/mL). In addition, nuclear transcription run-on assays typically demonstrated that HTG-VLDL increased PAI-1 gene transcription rates by ≈5- to 6-fold and ≈4- to 5-fold, respectively, primarily in the 2/2 PAI-1 genotype HUVEC cultures compared with 1/1 PAI-1 genotype HUVEC cultures or medium-incubated controls. The positive control interleukin-1 increased both 2/2 and 1/1 PAI-1 mRNA levels by ≈5- to 6-fold. Increased PAI-1 antigen and mRNA expression were associated with a concomitant 50% to 60% decrease in plasmin generation. These combined results demonstrate the genotype-specific regulation of PAI-1 expression by HTG-VLDL and Lp(a) and further indicate that these risk factor–associated components regulate PAI-1 gene expression at the transcriptional level in cultured HUVECs. Results from these studies further suggest that individuals with this responsive 2/2 PAI-1 genotype may reflect the additional inherent potential for later HTG-VLDL- or Lp(a)-induced fibrinolytic dysfunction, resulting in the early initiation of thrombosis, atherogenesis, and coronary artery disease.
Continued Thromboxane A
2
Formation Despite Administration of a Platelet Glycoprotein IIb/IIIa Antagonist in Patients Undergoing Coronary Angioplasty
Authors: Byrne Anthony , Moran Niamh , Maher Maureen , Walsh Noleen , Crean Peter , Fitzgerald Desmond J.
Published: June 2011

Abstract
Experimental data suggest that formation of thromboxane A
2
may be suppressed during administration of a glycoprotein IIb/IIIa antagonist. We determined the dose of one such compound, fradafiban, required to provide >80% occupancy of the platelet glycoprotein IIb/IIIa and examined its effects on thromboxane A
2
formation in patients undergoing PTCA. The dose response to fradafiban and additional effects of aspirin were explored initially in patients with stable coronary artery disease. Fradafiban induced a dose-dependent inhibition of platelet aggregation that correlated with fibrinogen receptor occupancy and plasma drug concentration. Addition of aspirin 300 mg had no effect on these parameters. At the highest dose, mean fibrinogen receptor occupancy was 89.7±1.2% (n=3) at 4 hours and platelet aggregation had decreased by 93.4±2.7%. Eighteen patients undergoing coronary angioplasty were randomized to receive either aspirin 330 mg or that dose of fradafiban producing >80% fibrinogen receptor occupancy. Platelet aggregation was suppressed throughout the infusion of fradafiban to a greater extent than with aspirin. However, there was a marked increase in urinary excretion of 11-dehydrothromboxane B
2
in patients treated with fradafiban: from 1973±889 to a peak of 9760±3509 pg/mg creatinine (
P
=.0046). Despite this evidence of continued platelet activation in vivo, there were no cases of coronary thrombosis. In conclusion, fradafiban suppresses platelet aggregation and may be a useful alternative to aspirin in the prevention of thrombotic events in patients undergoing PTCA. However, there is continued formation of thromboxane A
2
, which may continue to exert its effects as a potent vasoconstrictor and vascular smooth muscle mitogen.
Prostaglandin F2-Like Compounds, F2-Isoprostanes, Are Present in Increased Amounts in Human Atherosclerotic Lesions
Authors: Gniwotta Christina , Morrow Jason D. , Roberts L. Jackson , Kühn Hartmut
Published: June 2011

AbstractOxidative modification of LDL is believed to play a major role in atherogenesis. As major lipid peroxidation products oxygenated linoleic acid derivatives and oxysterols have been described in human atherosclerotic lesions. Here we report that human lesions contain isoprostanes as peroxidation products of arachidonic acid at a level of 27.1±21.2 pg/mg wet weight (n=10), which corresponds to 75.9±59.3 pg/mg dry weight. n contrast, human umbilical veins (n=10), which were used as nonatherosclerotic control vessels, contain much smaller amounts of isoprostanes (1.4±0.7 pg/mg wet weight, which corresponds to 11.7±6.2 pg/mg dry weight), and there are significant differences between the two types of vessels. As major products of linoleic acid oxidation, racemic hydroxy linoleate isomers were detected in the lesional ester lipids. In human lesions, the hydroxy linoleic acid/linoleic acid ratio was about 0.5%, a result indicating that 5 out of 1000 linoleate residues are present as hydroxylated derivatives. In umbilical veins, no hydroxy linoleic acid could be detected.

These data show that human atherosclerotic lesions contain increased amounts of hydroxy linoleic acid isomers and isoprostanes when compared with nonatherosclerotic vessel wall and suggest a link between local lipid peroxidation and progression of atherosclerosis. For evaluation of the degree of lipid peroxidation, the determination of the hydroxy linoleic acid/linoleic acid ratio appears to be more suitable than the isoprostane content.
Polymorphism of Angiotensin Converting Enzyme Gene Is Associated With Circulating Levels of Plasminogen Activator Inhibitor-1
Authors: Kim Duk-Kyung , Kim Jong-Won , Kim Seonwoo , Gwon Hyeon-Cheol , Ryu Jae-Choon , Huh Jeong-Eun , Choo Jin-A , Choi Youngran , Rhee Chong-Heon , Lee Won-Ro
Published: June 2011

Abstract
The deletion (D) allele of the insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene is strongly associated with an increased level of circulating ACE. The ACE gene polymorphism may influence the production of angiotensin II (Ang II). It has been shown that Ang II modulates fibrinolysis, that is, Ang II increases plasminogen activator inhibitor-1 (PAI-1) mRNA and plasma PAI-1 levels in vitro and in vivo. Considered together, we tested the hypothesis that the deletion allele of the ACE gene might be associated with increased levels of PAI-1. We related the
ACE
genotype to PAI-1 antigen levels in 603 men and 221 women attending a routine health screening. As a whole, the plasma PAI-1 level was not strongly associated with
ACE
genotype. Since the PAI-1 level was significantly influenced by well-known risk factors for coronary artery disease (CAD), we further analyzed the data after excluding subjects with major cardiovascular risk factors. In low-risk male subjects, the
DD
genotype had significantly higher levels of plasma PAI-1 (DD: 20.3±2.2; DI: 13.9±1.1; II: 13.6±1.3 ng/mL,
P
=.010 by ANOVA). In low-risk female subjects, the
DD
genotype showed a tendency to a high level of plasma PAI-1 without statistical significance. When analysis was restricted to postmenopausal women (age≥55 or FSH≥35 ng/mL), the
DD
genotype showed a significantly higher level of PAI-1 than subjects with the
DI
and
II
genotypes (27.7±6.2 versus 15.6±1.8 ng/mL,
P
=.028). The DD polymorphism of the ACE gene is associated with high PAI-1 levels in male and possibly in postmenopausal female subjects who have lower conventional cardiovascular risk factors. These results suggest that the increased ACE activity caused by DD polymorphism may play an important role in elevating the level of plasma PAI-1. Our data support the notion that the genetic variation of ACE contributes to the balance of the fibrinolytic pathway.
Insulin Resistance as an Independent Risk Factor for Carotid Artery Wall Intima Media Thickening in Vasospastic Angina
Authors: Shinozaki Kazuya , Hattori Yuichi , Suzuki Masaaki , Hara Yasushi , Kanazawa Akio , Takaki Hiroshi , Tsushima Motoo , Harano Yutaka
Published: June 2011

Abstract
Studies have shown the presence of insulin resistance together with compensatory hyperinsulinemia in vasospastic angina as well as obstructive coronary artery disease. There is growing evidence that the development of coronary atherosclerosis may be closely related to systemic atherosclerosis as well as coronary spasm. However, no information is available about the possible relationship between insulin resistance and the existence of carotid atherosclerosis in vasospastic angina without segmental stenosis or luminal irregularities in coronary angiograms. To evaluate the independent effect of insulin resistance on carotid intima media thickening, we performed insulin sensitivity tests (steady-state plasma glucose method) on 40 patients with vasospastic angina and 24 control subjects with angiographically intact coronary arteries. Both oral glucose tolerance tests and lipid analyses were performed. Using B-mode ultrasonography, we assessed intima media thickness and plaque formation of common carotid arteries in these subjects. The steady-state plasma glucose level in the vasospastic angina group was about twofold higher than that of the control group, confirming the presence of insulin resistance in patients with vasospastic angina. The patients with vasospastic angina showed a significant increase in the average intima media thickness of the carotid wall and frequency of plaque formation, although they were comparable to the control subjects in risk factors other than insulin resistance. The intima media thickness was correlated with age (
r
=.62,
P
<.001), 2-hour insulin area (
r
=.45,
P
<.01), and steady-state plasma glucose level (
r
=.68,
P
<.0001) in patients with vasospastic angina. Similar correlations were observed in the control subjects. Multiple regression analyses of data indicated that 67% of the variation in the intima media thickness could be accounted for by age, steady-state plasma glucose level, and cigarette-years in vasospastic angina. In addition, differences in IMT were independently related to vasospastic angina. These results suggest that insulin resistance in association with compensatory hyperinsulinemia may be an important pathogenic factor for the development of coronary artery spasms and systemic early atherosclerosis.
Neonatal Diagnosis of Familial Hypercholesterolemia in Newborns Born to a Parent With a Molecularly Defined Heterozygous Familial Hypercholesterolemia
Authors: Vuorio Alpo F. , Turtola Hannu , Kontula Kimmo
Published: June 2011

Abstract
This study was designed to compare blood lipid levels in newborn individuals with molecularly defined heterozygous familial hypercholesterolemia [FH] to those in nonaffected babies and to clarify the value of lipid determinations in assessment of diagnosis of FH at birth and 1 year of age. Twenty-five babies were born to 21 parents with DNA-documented heterozygous FH. Analysis of their cord blood samples revealed 11 newborns with the FH-North Karelia [FH-NK] mutation, 3 newborns with the FH-Helsinki [FH-HKI] mutation, and 11 nonaffected newborns. Cord serum total [TC] and LDL cholesterol [LDL-C] levels (mean±SD) in affected newborns (2.60±0.70 and 1.77±0.56, respectively) were significantly (
P
<.001) higher than those in nonaffected ones (1.54±0.23 and 0.78±0.15, respectively) and another cohort of 30 randomly selected control samples from apparently healthy newborns (1.84±0.46 and 1.03±0.30, respectively). However, there was overlapping of individual lipid levels in these three groups precluding the use of TC or LDL-C determinations in neonatal diagnosis of FH. In contrast, 1 year follow-up samples from 10 affected and 7 nonaffected individuals, as well as additional samples collected from another group of 8 affected and 9 nonaffected individuals, indicated that serum cholesterol levels showed much greater increment in children with FH. Thus, at the age of 1 year the mean serum TC and LDL-C levels in the affected infants (8.38±1.18 and 7.02±1.07, respectively) were much higher (
P
<.001) than the corresponding levels (4.40±0.66 and 2.89±0.68, respectively) in the nonaffected infants, and the individual ranges of TC and LDL-C levels were nonoverlapping in these two groups. Serum HDL cholesterol [HDL-C] levels in 1-year-old children with FH (0.95±0.14) were approximately 20% lower than those of their nonaffected counterparts (1.16±0.15,
P
<.001), after being similar at birth. In conclusion, phenotypic expression of heterozygous FH, as defined by molecular analysis of genomic DNA, is evident in serum LDL-C (but not HDL-C) levels already at birth, but for diagnostic purposes blood lipid determinations carried out at the age of 1 year are highly superior to those performed at birth.
Demonstration of Biphasic Effects of Docosahexaenoic Acid on Apolipoprotein B Secretion in HepG2 Cells
Authors: Wu Xujun , Shang Aiming , Jiang Hongshi , Ginsberg Henry N.
Published: June 2011

Abstract
Oleic acid (OA) stimulates apolipoprotein B (apoB) secretion from HepG2 cells by protecting the nascent protein from rapid intracellular degradation. In contrast, the n-3 fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid, have been shown to reduce apoB secretion by increasing its intracellular degradation in rat hepatocytes. We attempted to determine if OA and DHA have these opposite effects at the same point in the secretory pathway for apoB or if they act at different points in HepG2 cells. Unexpectedly, we found that when DHA (0.2 mmol/L) was incubated with HepG2 cells for 2 hours, it stimulated both triglyceride (TG) synthesis and apoB secretion significantly (the “stimulatory effect”). The stimulatory effect of DHA on apoB secretion was associated with decreased intracellular degradation of newly synthesized apoB. These acute effects of DHA on TG synthesis and apoB secretion paralleled those previously demonstrated with OA. After DHA was removed from the medium, however, both TG synthesis and apoB secretion rapidly decreased to a level that was significantly less than the control level (the “inhibitory effect”). At the same time, intracellular apoB degradation was significantly increased, and this degradation was efficiently prevented by proteasome inhibitors. Removal of DHA from the incubation resulted in inhibition of the incorporation of endogenous fatty acids into TG. In contrast, removal of OA from the media was not associated with any such inhibitory effect. The inhibitory effect of DHA on basal apoB secretion persisted at least 8 hours. These studies suggest that incubation of HepG2 cells with DHA has biphasic effects on TG synthesis and apoB secretion: an initial stimulation of TG synthesis is followed by inhibition of TG synthesis and increased apoB degradation. Although the stimulatory effect of DHA is apparent during short incubations of HepG2 cells, both effects would be expected to occur during long incubations, since fatty acid uptake by cells is rapid and efficient. Thus, long incubations of HepG2 cells with DHA could result in overall reduced apoB secretion compared with cells incubated in bovine serum albumin. If these findings are extrapolated to the in vivo situation, they can explain the ability of dietary n-3 fatty acids, which would be delivered to the liver intermittently, to reduce very low density lipoprotein secretion.
Effect of Atherosclerosis on Transmural Convection and Arterial Ultrastructure
Authors: Baldwin Ann L. , Wilson Lisa M. , Gradus-Pizlo Irmina , Wilensky Robert , March Keith
Published: June 2011

Abstract
Local infusion of agents through perforated catheters may reduce neointimal formation following vascular angioplasty. Such treatment will succeed only if the drug is retained within the arterial intima long enough to promote repair. Drugs will be dispersed throughout the wall predominantly by transmural convection instead of diffusion if the Peclet number, Pe=J(1-ς
f
)/P, is greater than unity, where J is the transmural fluid flow per unit surface area and ς
f
and P are the reflection and permeability coefficients to the drug, respectively. Although the targets of local drug delivery will be atherosclerotic vessels, little is known about the transport properties of these vessels. Accordingly, we evaluated the effects of hypercholesterolemia and atherosclerosis on J per unit pressure (hydraulic conductance, L
p
) and on ultrastructure in femoral arteries. Measurements were made at 30, 60, and 90 mm Hg in anesthetized New Zealand white rabbits fed a normal diet (n=6) and after 3 weeks of lipid feeding (n=19). Atherosclerosis was induced in six lipid-fed animals by air desiccation of a femoral artery. Hydraulic conductance was significantly greater in vessels from hypercholesterolemic than from normal animals and decreased with pressure only in hypercholesterolemic arteries. Atherosclerosis did not augment hydraulic conductance compared with hypercholesterolemia alone. Electron microscopic examination demonstrated damaged endothelium in hypercholesterolemic arteries and both altered endothelium and less tightly packed medial tissue, compared with controls, in atherosclerotic vessels, at least at lower pressures. Peclet numbers for macromolecules exceeded unity for all three groups of arteries and reached 0.3 to 0.4 for molecules as small as heparin. Thus, convection plays a dominant role in the distribution of macromolecular agents following local delivery and may result in their rapid transport to the adventitia in the femoral artery.
Evidence for Hormone-Sensitive Lipase mRNA Expression in Human Monocyte/Macrophages
Authors: Reue Karen , Cohen Robert D. , Schotz Michael C.
Published: June 2011

AbstractThe role of hormone-sensitive lipase (HSL) in the hydrolysis of adipose tissue triacylglycerol to provide free fatty acids for energy requirements has been well established. However, the role of HSL in other tissues, including macrophages, is not well understood. The demonstration that HSL is capable of hydrolyzing cholesteryl esters at approximately the same rate as triacylglycerol raised the possibility that HSL activity in macrophages may influence the accumulation of cholesteryl esters in foam cells of atherosclerotic lesions. We and others have previously demonstrated that HSL mRNA is expressed in murine peritoneal macrophages and macrophage cell lines; however, it was previously reported that HSL mRNA is absent in human monocyte-derived macrophages, suggesting that a species difference may exist. To clarify this point, we have further examined the issue of HSL mRNA expression in human macrophages. In the current study, we demonstrate that HSL mRNA is detectable in human monocyte-derived macrophages and in the THP-1 human monocyte cell line using reverse transcription coupled to polymerase chain reaction (RT-PCR). Specific amplification of cDNA derived from mRNA was ensured by using primers that span an intron within the human HSL gene, and the identity of PCR products as HSL was confirmed by hybridization to HSL cDNA and by DNA sequencing. Using a semiquantitative PCR assay, we establish that HSL mRNA levels in monocyte/macrophages are approximately 1/40 the levels in human adipose tissue. These results indicate that further studies addressing the role of HSL in macrophage metabolism and its potential role in development of foam cells in human atherosclerotic lesions are warranted.
Association of Apolipoprotein Genetic Polymorphisms With Plasma Cholesterol in a Japanese Rural Population
Authors: Zaman Mohammad Mostafa , Ikemoto Shinji , Yoshiike Nobuo , Date Chigusa , Yokoyama Tetsuji , Tanaka Heizo
Published: June 2011

AbstractThe association between apolipoprotein (apo) genetic polymorphisms and lipid phenotypes remains to be determined because such studies have reported contradictory results. We have measured plasma total cholesterol (TC) and HDL cholesterol (HDL-C) in a cross-sectional sample of 1328 (462 men and 866 women) Japanese (aged 40 to 80 years) and analyzed their DNA for the following genotypes: apoA1-C3MspI andSstI sites; apoB signal peptide insertion/deletion,XbaI site and 3′ variable number of tandem repeats (VNTR); and apoE. Using multivariate analyses (ANOVA) we found that (1) the polymorphisms of apoA1-C3 and apoB genes are not significantly associated with TC or HDL-C and (2) the polymorphism of the apoE gene is significantly related with TC and the TC:HDL-C ratio. The ε2 carriers have lower levels of TC and a lower TC:HDL-C ratio, ε3 carriers have intermediate levels, and ε4 carriers have higher levels. These findings held whether sexes were analyzed separately or together. Although an opposite trend in HDL-C levels was observed, it did not reach the level of statistical significance. Multiple regression analyses have shown that apoE polymorphism accounts for about 2.3% of the variation in TC and TC:HDL-C ratio irrespective of sex. When the subjects are divided into two groups according to their age (≤59 and ≥60 years old), the effect of apoE alleles on serum cholesterols appears to be diluted in the younger age group whereas it appears to be accentuated in the older group for both sexes. Our findings suggest that among the apo genetic polymorphisms examined, the apoE gene may be considered as a possible candidate for the “high-risk” strategy of atherosclerosis prevention in the Japanese population.
Copper-Catalyzed Oxidation Mediates PAF Formation in Human LDL Subspecies
Authors: Tsoukatos Demokritos C. , Arborati Muriel , Liapikos Theodoros , Clay Keith L. , Murphy Robert C. , Chapman M. John , Ninio Ewa
Published: June 2011

AbstractFree radical-mediated oxidation of cholesterol-rich LDL plays a key role in atherogenesis and involves the formation of oxidized phospholipids with proinflammatory biological activity. We evaluated the production of platelet-activating factor (PAF), a potent inflammatory mediator, in human LDL subspecies on copper-initiated oxidation (4 μmol/L CuCl2, 80 μg/mL for 3 hours at 37°C). PAF formation was determined by biological assay of HPLC-purified lipid extracts of copper-oxidized lipoproteins; chemical identity was confirmed by gas chromatographic and mass spectrometric analyses. PAF, characterized as the C16:0 molecular species, was preferentially produced in intermediate LDL (d=1.029 to 1.039 g/mL) (8.6±5.7 pmol PAF/3 h per mg LDL protein) and light LDL (d=1.019 to 1.029 g/mL), but was absent from dense LDL particles (d=1.050 to 1.063 g/mL). As PAF:acetylhydrolase inactivates PAF and oxidized forms of phosphatidylcholine, we evaluated the relationship of lipoprotein-associated PAF:acetylhydrolase to PAF formation. We confirmed that PAF:acetylhydrolase activity was elevated in native, dense LDL (41.5±9.5 nmol/min per mg protein) but low in LDL subspecies of light and intermediate density (d 1.020 to 1.039 g/mL) (3.5±1.6 nmol/min per mg protein) [Tselepis et al,Arterioscler Thromb Vasc Biol.1995;15:1764–1773]. On copper-mediated oxidation for 3 hours at 37°C, dense LDL particles conserved 20±14% of their initial enzymatic activity; in contrast, PAF:acetylhydrolase activity was abolished in light and intermediate LDL subspecies. Clearly, the elevated PAF: acetylhydrolase activity of dense LDL efficiently diminishes the potential inflammatory role of endogenously formed PAF; nonetheless, formation of proatherogenic lysophospholipids results. In contrast, LDL particles of the light and intermediate subclasses can accumulate PAF on oxidative modification.
Impact of Gender on the Metabolism of Apolipoprotein A-I in HDL Subclasses LpAI and LpAI:AII in Older Subjects
Authors: Tilly-Kiesi Marju , Lichtenstein Alice H. , Joven Jorge , Vilella Elisabet , Cheung Marian C. , Carrasco Wanda V. , Ordovas Jose M. , Dolnikowski Gregary , Schaefer Ernst J.
Published: June 2011

Abstract
The behavior of apolipoprotein (apo) A-I in lipoprotein (Lp) AI and LpAI:AII was studied in 11 postmenopausal females and 11 males matched for plasma triglyceride and total cholesterol levels. Subjects consumed a baseline diet [35% fat (14% saturated, 15% monounsaturated, and 7% polyunsaturated), 15% protein, 49% carbohydrate, and 147 mg cholesterol/1000 kcal] for 6 weeks before the start of the kinetic study. At the end of the diet period, using a primed-constant infusion of [5,5,5-
2
H
3
]leucine, residence times (RT) and secretion rates (SR) of apoA-I were determined in 2 subpopulations of high-density lipoprotein (HDL) particles, LpAI and LpAI:AII. Plasma total cholesterol, low-density lipoprotein cholesterol, and triglyceride concentrations were similar in males and females. The mean plasma HDL cholesterol concentration in males (1.14± 0.23 mmol/L; mean±SD) was lower than in females (1.42±0.18 mmol/L;
P
=.0034). Similarly, the mean plasma concentration of apoA-I in males (130±21 mg/dL) was lower than that in females (150±19 mg/dL;
P
=.0421). The RT of apoA-I in either LpAI or LpAI:AII was similar between men and women. Despite the higher plasma apo A-I levels in female compared with male subjects, total apoA-I and apoA-I in LpAI and LpAI:AII pool sizes were similar between the two groups, attributable to the lower body weight of the female subjects. The mean SR of total apoA-I in males (8.5±2.7 mg · kg
−1
· d
−1
) was 22% lower than in females (10.9±2.3 mg · kg
−1
· d
−1
;
P
=.0389). The SR of both apoA-I in LpAI and LpAI:AII was lower in males than females, although the differences did not reach statistical significance. These data suggest that the difference observed in HDL cholesterol concentration between males and females is attributable to SR of apoA-I and not the catabolic rate.
Hypertensive Nephropathy and the Gene for Angiotensin-Converting Enzyme
Authors: Kario Kazuomi , Kanai Nobuyuki , Nishiuma Shinichi , Fujii Takeshi , Saito Ken , Matsuo Takefumi , Matsuo Masafumi , Shimada Kazuyuki
Published: June 2011

To investigate the genetic determinants for microalbuminuria, we studied an insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene, which influences the plasma ACE level, in 333 consecutive hypertensive patients and 113 normotensive control subjects. The urinary albumin excretion rate was calculated by using a 12-hour urine collection (mean for two consecutive nights from 7
pm
to 7
am
) in all 333 hypertensive patients. The ACE D allele frequency did not differ significantly between the hypertensive patients and the normotensive control subjects (0.37 and 0.33, respectively). Among the hypertensive patients, nephropathy (microalbuminuria and albuminuria) was more common (
P
<.001) in those with the ACE DD genotype than in those with other genotypes. The D allele frequency in the nephropathy group was significantly higher than that in the normoalbuminuric group (0.45 versus 0.32, χ
2
=10.8,
P
<.001). These results indicate that ACE I/D polymorphism is a genetic determinant for hypertensive renal disease in hypertensive patients. This polymorphism might be a confounding factor involved in the association between hypertensive nephropathy and cardiovascular events.
Macrophages Isolated From Human Atherosclerotic Plaques Produce IL-8, and Oxysterols May Have a Regulatory Function for IL-8 Production
Authors: Liu Yani , Hultén Lillemor Mattsson , Wiklund Olov
Published: June 2011

Oxysterols are biologically active molecules generated during oxidation of LDL. Several of these oxysterols were found in macrophage-derived foam cells from human atherosclerotic tissue (eg, 7-hydroxycholesterol, 7-ketocholesterol, 5-epoxycholesterol, and 25-hydroxycholesterol). A specific stimulation of interleukin-8 (IL-8) production by oxidized LDL (oxLDL) has been shown by other investigators. In foam cells from human atherosclerotic tissue, we found high levels of IL-8 (183.1 pg/10
6
cells) compared with monocytes (23.2 pg/10
6
cells) or monocyte-derived macrophages in culture (1.5 pg/10
6
cells). When monocytes and monocyte-derived macrophages, in vitro, were exposed to a series of different oxysterols, we found that all oxysterols tested had a tendency to stimulate IL-8 production but that 25-hydroxycholesterol was the most potent one. This stimulation of IL-8 production was time and dose dependent and could be blocked by cycloheximide. These results indicate that oxysterols in oxLDL may have a regulatory effect on IL-8 production. IL-8, a potent chemoattractant, may play a role in the recruitment of T lymphocytes and smooth muscle cells into the subendothelial space and may contribute to the formation of atherosclerotic lesions.
Effect of 17β-Estradiol on Plasma Lipids and LDL Oxidation in Postmenopausal Women With Type II Diabetes Mellitus
Authors: Brussaard Hanny E. , Leuven Jan A. Gevers , Kluft Cornelis , Krans H. Michiel J. , Duyvenvoorde Wim van , Buytenhek Rien , Laarse Arnoud van der , Princen Hans M.G.
Published: June 2011

In type II diabetes mellitus the altered hormonal state after menopause may represent an additional cardiovascular risk factor. Estrogen replacement therapy (ERT) is associated with a decreased cardiovascular risk, at least in nondiabetic postmenopausal women. We studied the effect of ERT on plasma lipids and lipoproteins and on LDL oxidation in 40 postmenopausal women with type II diabetes but with minimal vascular complications in a randomized placebo-controlled trial. Twenty patients were treated orally with 2 mg/d micronized 17β-estradiol and 20 patients with placebo for 6 weeks. Plasma total cholesterol (−6%,
P
=.04), LDL cholesterol (−16%,
P
=.0001), and apoB (−11%,
P
=.001) levels decreased and HDL cholesterol (20%,
P
=.0001) and apoA-I (14%,
P
=.0001) levels increased after ERT compared with placebo. Glycated hemoglobin (HbA1c) decreased significantly after ERT (−3%,
P
=.03), the cholesterol content of the LDL particles decreased (−5%,
P
=.006), triglyceride content increased (16%,
P
=.01), and LDL particle size did not change significantly. ERT had no effect on parameters of LDL oxidation. We conclude that plasma levels of HDL cholesterol, apoA-I, LDL cholesterol, apoB, and glycated hemoglobin are improved in postmenopausal women with type II diabetes mellitus after treatment with 17β-estradiol, indicative of a better metabolic control, and that ERT has no effect on LDL oxidizability.
Oxidized LDLs Induce Massive Apoptosis of Cultured Human Endothelial Cells Through a Calcium-Dependent Pathway
Authors: Escargueil-Blanc Isabelle , Meilhac Olivier , Pieraggi Marie-Thérèse , Arnal Jean-François , Salvayre Robert , Nègre-Salvayre Anne
Published: June 2011

Oxidized LDLs are thought to play a central role in atherogenesis. Among their wide variety of biological properties, oxidized LDLs exhibit a cytotoxic effect on cultured vascular cells. Toxic doses of mildly oxidized LDLs elicited massive apoptosis in both primary and immortalized cultures of endothelial cells as shown by characteristic morphological and biochemical changes. Cytoplasmic and nucleic modifications (eg, chromatin condensation and nucleus fragmentation) were visualized by using electron and fluorescence microscopy of intact cells labeled by the fluorescent DNA probe SYTO-11. DNA fragmentation was quantified by ultracentrifugation of chromatin fragments, evaluated in situ by using the TUNEL (Terminal transferase-mediated dUTP-biotinnickendlabeling) procedure, and visualized by electrophoresis of radiolabeled DNA fragments showing the characteristic apoptotic ladder. Apoptotic cells became rapidly detached and underwent postapoptotic necrosis that led to cell disintegration. Apoptosis was subsequent to a sustained and delayed peak of cytosolic calcium. Both the calcium peak and apoptosis were blocked by chelating the extracellular calcium with EGTA or by inhibiting the calcium influx by the calcium-channel blockers nifedipine and nisoldipine, thus suggesting that the apoptotic process induced by oxidized LDLs is clearly calcium dependent. Aurintricarboxylic acid, an inhibitor of endonucleases, also blocked the apoptotic process without blocking the calcium peak. These results suggest that toxic doses of mildly oxidized LDLs induce massive apoptosis of endothelial cells through a calcium-dependent mechanism and that this apoptotic process can be prevented by inhibiting the rise of cytosolic calcium or by inhibiting cellular endonucleases by aurintricarboxylic acid.
Homozygous Familial Defective Apolipoprotein B-100
Authors: Schaefer Juergen R. , Scharnagl Hubert , Baumstark Manfred W. , Schweer Horst , Zech Loren A. , Seyberth Hansjörg , Winkler Karl , Steinmetz Armin , März Winfried
Published: June 2011

Familial defective apolipoprotein B-100 (FDB) is a frequently inherited disorder of lipoprotein metabolism. The glutamine-for-arginine substitution at position 3500 of apolipoprotein (apo) B-100 leads to defective binding of apo B-100 to the low density lipoprotein (LDL) receptor and accumulation of LDL in the plasma. We recently identified a patient homozygous for this mutation. His LDL cholesterol and apo B concentrations were approximately twice normal, whereas his apo E plasma level was low. Using a stable-isotope labeling technique ([
2
H
3
]leucine–primed constant infusion), we studied lipoprotein turnover in vivo in the fasting state in this patient and three clinically healthy, normolipidemic individuals not carrying the FDB mutation. The residence time of LDL apo B-100 was prolonged 3.6-fold in the FDB homozygote (8.3 vs 2.3 days). The production rate of LDL apo B-100 was decreased (7.4 vs 15 mg per kg per day). In FDB the residence time of very low density lipoprotein (VLDL) apo B-100 was longer (2.6 vs 1.3 hours), whereas the residence time of VLDL apo E was shorter (2.6 vs 4.5 hours) than normal. These data show that the in vivo metabolism of apo B-100–containing lipoproteins in FDB is different from that in familial hypercholesterolemia, in which LDL receptors are defective. In both conditions the residence times of LDL apo B-100 appear to be increased to approximately the same degree. This contrasts with the LDL apo B-100 synthetic rate, which is increased in familial hypercholesterolemia and decreased in FDB. The decreased production of LDL apo B-100 in FDB may originate from enhanced removal of apo E–containing LDL precursors by LDL receptors, which may be upregulated in response to the decreased flux of LDL-derived cholesterol into hepatocytes.
Early Activation Signals in Endothelial Cells
Authors: Introna Martino , Mantovani Alberto
Published: June 2011

AbstractWith limitation to the “proinflammatory program” induced in endothelial cells by exposure to interleukin-1, tumor necrosis factor, and interleukin-6, we review the available data on the signaling for these three cytokines, from receptor engagement to induction of gene transcription. Only a few molecular pathways have been characterized so far, and key issues in endothelial biology, such as endothelial specificity of gene expression and heterogeneity of different endothelial populations, remain largely unexplored.
Vitamin E Inhibits Low-Density Lipoprotein–Induced Adhesion of Monocytes to Human Aortic Endothelial Cells In Vitro
Authors: Martin Antonio , Foxall Thomas , Blumberg Jeffrey B. , Meydani Mohsen
Published: June 2011

AbstractMonocyte adhesion to human aortic endothelial cells (ECs) is one of the early events in the development of atherogenesis. ECs were used to investigate the role of vitamin E in human monocyte adhesion to ECs in vitro. ECs incubated with 40 to 193 mg/dL of low-density lipoprotein cholesterol (LDL) for 22 hours exhibited increasing dose-dependent adherence for untreated, isolated human monocytes (P<.05). ECs exposed to the highest dose of LDL (193 mg/dL) but pretreated with 19 μmol/L α-tocopherol for 24 hours showed a trend to lower adherence for monocytes compared with nontreated ECs (4.4±1.2% versus 7.6±1.9%;P=.09). This effect of vitamin E became more significant (P<.05) when ECs were exposed to a lower level of LDL (40 mg/dL) or were pretreated with a higher level of α-tocopherol (42 μmol/L) and then exposed to 80 mg/dL LDL. Presupplementation of ECs with 15, 19, and 37 μmol/L α-tocopherol significantly (P<.05) reduced monocyte adhesion by 6±1%, 37±6%, and 69±17%, respectively. Levels of soluble intercellular adhesion molecule-1 (sICAM-1), one of the adhesion molecules for monocytes, increased after incubation of ECs with LDL 80 mg/dL (4.7±0.7 versus 6.4±1.2 ng/mL, respectively;P<.05). Treatment of ECs with α-tocopherol (42 μmol/L) significantly reduced induction of sICAM-1 by LDL to 2.2±2.3 ng/mL. After exposure to LDL, prostaglandin I2production by ECs was diminished, whereas presupplementation of ECs with α-tocopherol partially reversed the LDL effect. Production of interleukin-1β was not detectable when ECs were treated with α-tocopherol, LDL, or α-tocopherol followed by LDL. Our findings indicate that vitamin E has an inhibitory effect on LDL-induced production of adhesion molecules and adhesion of monocytes to ECs via its antioxidant function and/or its direct regulatory effect on sICAM-1 expression.
Thrombin Promotes Activation of Matrix Metalloproteinase-2 Produced by Cultured Vascular Smooth Muscle Cells
Authors: Galis Zorina S. , Kranzhöfer Roger , Fenton John W. , Libby Peter
Published: June 2011

Abstract
Thrombin generated at sites of vascular injury not only participates in the coagulation cascade but can signal other events related to development and complication of atherosclerotic plaques. We investigated here a novel nonthrombotic action of thrombin: the possibility that this protease influences the expression or activation of matrix metalloproteinases (MMPs) produced by vascular smooth muscle cells (SMCs). Matrix-degrading proteinases likely contribute to several aspects of vascular lesion development. Vascular SMCs constitutively elaborate the zymogen form of gelatinase A (MMP-2), found in cell supernatants complexed with its inhibitor, the tissue inhibitor of metalloproteinases (TIMP)-2. When activated, MMP-2 digests collagens and elastin and may thus promote cell migration and vascular remodeling. Analysis of culture supernatants harvested from either human or rabbit vascular SMCs by gelatin zymography revealed that compared with supernatants of unstimulated SMCs, media conditioned by thrombin-stimulated cells contained increased amounts of proteolytically processed MMP-2, suggesting activation of this MMP. Further experiments tested whether thrombin directly activates MMP-2. In cell-free experiments, when added to medium harvested from unstimulated SMCs, α-thrombin increased in a dose- and time-dependent manner the amount of proteolytically processed MMP-2, as shown by zymography and by Western blotting with specific antibodies. Thrombin cleaved pro–MMP-2 within 4 hours, even when the gelatinase was bound with its inhibitor, TIMP-2. Thrombin treatment rendered culture media of unstimulated SMCs able to degrade collagen type IV, consistent with generation of active MMP-2. Addition of inhibitors of either thrombin or MMPs decreased this type IV collagenolytic activity, but thrombin in the absence of SMC-conditioned medium containing pro–MMP-2 exhibited only minimal collagenolysis. Our results suggest that at sites of vascular injury, thrombin may activate locally produced MMP-2 and thereby facilitate cell migration and proliferation. In the case of complicated atherosclerotic plaques, episodes of intraplaque hemorrhage or plaque disruption with thrombosis may promote plaque instability by increasing local matrix-degrading activity.
Circulating Vascular Cell Adhesion Molecule-1 Correlates With the Extent of Human Atherosclerosis in Contrast to Circulating Intercellular Adhesion Molecule-1, E-Selectin, P-Selectin, and Thrombomodulin
Authors: Peter Karlheinz , Nawroth Peter , Conradt Christian , Nordt Thomas , Weiss Thomas , Boehme Michael , Wunsch Andreas , Allenberg Jens , Kübler Wolfgang , Bode Christoph
Published: June 2011

Abstract
Secondary prevention of atherosclerosis, especially before the onset of symptoms, appears desirable and could be possible with a serum marker detecting atherosclerosis. Circulating, shedded forms of adhesion molecules may serve as such because their expression is upregulated in atherosclerotic plaques. In 52 patients with peripheral arterial vascular disease (Fontaine class IIa, 7 patients; class IIb, 29 patients; and class III, 16 patients), the extent of atherosclerosis was evaluated on the basis of angiograms of a large portion of the arterial system. The area diseased by atherosclerosis was determined by the percentage of vessel wall irregularities of the following calculated segments: aorta (distal from the kidney arteries), common iliac artery, external iliac artery, common femoral artery, lateral circumflex femoral artery, and popliteal artery. The maximal surface area that could exhibit atherosclerotic changes was 250 cm
2
. The serum concentration of circulating vascular cell adhesion molecule-1 (VCAM-1) correlated with the extent of atherosclerosis (
r
=.8,
P
<.001). In contrast, circulating intercellular adhesion molecule-1, E-selectin, P-selectin, and thrombomodulin (as markers for endothelial cell damage) did not correlate with the extent of atherosclerosis. Furthermore, circulating VCAM-1 could be used to indicate stages of atherosclerosis with a high degree of statistical significance. The potential bias of factors such as age, diabetes mellitus, hypercholesterolemia, arterial hypertension, renal failure, and history of myocardial infarction on the correlation of circulating VCAM-1 with the extent of atherosclerosis could be excluded by multivariate analysis. These findings suggest an important role of VCAM-1 in atherosclerosis and may serve as the basis for further evaluation of circulating VCAM-1 as a potential serum marker for atherosclerosis.
Epitope Specificity of Anti–Heat Shock Protein 65/60 Serum Antibodies in Atherosclerosis
Authors: Metzler Bernhard , Schett Georg , Kleindienst Roman , van der Zee Ruurd , Ottenhoff Tom , Hajeer Ali , Bernstein Robert , Xu Qingbo , Wick Georg
Published: June 2011

AbstractLevels of specific antibodies (Ab) against mycobacterial and human heat shock protein (hsp) 65/60 are increased in the sera of patients with atherosclerotic lesions and have been demonstrated to be capable of mediating endothelial cytotoxicity. To clarify the antigen epitopes recognized by these serum Abs, Ab binding to hsp65 deletion mutants (Dms), as well as to overlapping 15-mer and 8-mer hsp65 peptides, was assessed. Western blotting of hsp65 Dms indicated the presence of at least one epitope between amino acid (aa) residues 171and 276, recognized by both high-titer sera and affinity-purified anti-hsp65/60 Ab. Fluorescence immunoassays using 53 15-mer peptides and Pin ELISA using 526 7-mer peptides demonstrated three distinct, conserved sequences with high affinity to high-titer sera and purified anti-hsp65/60 Ab. TwoN-terminal sequences, aa 97-109 and aa 179-187, and oneC-terminal sequence, aa 504-512, were identified. These three epitopes recognized by anti-hsp65/60 Ab may serve as autoantigens in certain circumstances in vivo. This phenomenon could contribute to the initiation of atherosclerosis by an autoimmune reaction.
Manipulating Angiogenesis
Authors: Pepper Michael S.
Published: June 2011

Abstract
Considerable progress has been made recently in understanding the molecular mechanisms of angiogenesis, which like most other biological processes is the result of subtle and often complex interactions between molecules that have regulatory (eg, cytokines and their receptors) and effector (eg, extracellular matrix, integrins, and proteases) functions. The title of this review was chosen to reflect a recent trend in which knowledge acquired through a molecular/cell biological approach is being rapidly transferred to the clinical setting. As a result, by manipulating angiogenesis either positively or negatively, considerable therapeutic benefit can now be envisaged in physiological and pathological settings in which neovascularization is a prominent component.
Relationship Between Serum Sex Hormones and Coronary Artery Disease in Postmenopausal Women
Authors: Phillips Gerald B. , Pinkernell Bruce H. , Jing Tian-Yi
Published: June 2011

Abstract
Although sex hormones appear to be importantly involved in the development of coronary heart disease, apparently no study has yet reported an alteration in an endogenous sex hormone level in relation to coronary heart disease in women. In an attempt to determine whether any sex hormone abnormality might be a factor in the development of myocardial infarction in women, estradiol and testosterone, as well as sex hormone–binding globulin, insulin, dehydroepiandrosterone sulfate, and risk factors for myocardial infarction, were measured in relation to the degree of coronary artery disease (CAD) in 60 postmenopausal women undergoing coronary angiography. In a multiple-regression analysis with the degree of CAD as the dependent variable and free testosterone (FT), estradiol, age, body mass index, systolic blood pressure, cholesterol, smoking, and insulin as independent variables in the model, only FT (
P
<.008) and cholesterol (
P
=.01) were significantly related to the degree of CAD, both positively. To exclude a possible confounding effect due to prior myocardial infarction, the multiple-regression analysis was repeated for the subgroup of 49 patients remaining after excluding the 11 patients who had ever had a myocardial infarction; again only FT (
P
<.04) and cholesterol (
P
=.05) were significantly related to the degree of CAD. Neither total testosterone in place of FT nor HDL cholesterol in place of total cholesterol in the model was significantly related to CAD. Sex hormone–binding globulin and dehydroepiandrosterone sulfate, added individually to the model, showed no significant relationship to CAD. These results raise the possibility that in women an elevated FT level may be a risk factor for coronary atherosclerosis.