bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Factor XIa–α
1
-Antitrypsin in Plasma, a Contact Phase–Activated Coagulation Factor–Inhibitor Complex, in Patients With Coronary Artery Disease
Authors: Murakami Takashi , Komiyama Yutaka , Masuda Midori , Karakawa Masahiro , Iwasaka Toshiji , Takahashi Hakuo
Published: June 2011

Abstract
Excess activated factor XI (FXIa) in plasma indicates increased activation during the contact phase of blood coagulation. To investigate the relationship between such elevations and coronary atherosclerosis, we examined FXIa values in patients with coronary artery disease (CAD) by an enzyme-linked immunorsorbent assay method that we developed that detects FXIa in plasma samples as an FXIa–α
1
-antitrypsin complex (FXIa-α
1
AT). The presence and extent of CAD were documented by coronary angiography and assessed by a recently developed scoring system for semiquantitative estimation of coronary atherosclerosis. Plasma FXIa-α
1
AT levels were significantly increased in patients with angiographically proven CAD (13.9±3.0 μg/L, n=42) compared with age-matched, healthy control subjects (11.9±1.7 μg/L, n=20) as well as patients with angiographically normal coronary arteries (12.0±2.3 μg/L, n=25). Moreover, in the total patient population, the FXIa-α
1
AT level was related to the number of significant coronary artery stenoses as well as to the total coronary score. FXIa-α
1
AT showed a positive correlation with thrombin–antithrombin III complex, fibrinogen, and Lp(a) and an inverse correlation with apo A-I, as determined by multivariate analysis. Our studies provide evidence that increased activation of the contact pathway occurs in patients with CAD and is related to the severity of the disease. Although it is unknown whether this abnormality is the cause or the result of the vascular lesion, it may be important for progression of the underlying atherosclerosis or for propagation of the atherosclerotic process itself.
Tissue Factor Pathway Inhibitor Activity Associated With LDL Is Inactivated by Cell- and Copper-Mediated Oxidation
Authors: Lesnik Philippe , Dentan Christine , Vonica Alin , Moreau Martine , Chapman M. John
Published: June 2011

AbstractHuman plasma contains a multivalent, Kunitz-type proteinase inhibitor termed tissue factor pathway inhibitor (TFPI), which is a specific inhibitor of the action of the factor VII(a)–tissue factor complex in coagulation. A major fraction of plasma TFPI is transported in association with LDL. Because LDL may undergo oxidation in the arterial wall during atherogenesis, we examined the effect of copper- and cell-mediated oxidative modification on TFPI activity associated with LDL. Oxidation mediated by copper ions resulted in a significant inactivation of LDL-associated TFPI (60% to 72% at 24 hours with 2.5 μmol/L CuCl2). The inactivation of TFPI was strongly negatively correlated with both an increase in the net electrical charge of LDL (r=−.80,P≤.0001) and with the production of thiobarbituric acid–reactive substances (r=−.78,P≤.0001) and lipid peroxides (r=−.80,P≤.0001). Cell-mediated oxidation, involving incubation of LDL for 48 hours with either monocyte-like THP1 cells or human monocytes in Ham’s F-10 medium, effected a significant decrease (64% and 75%, respectively) in LDL-associated TFPI activity. By contrast, prolonged exposure of LDL to purified soybean lipoxygenase (5000 U/mL) was less effective in inactivating TFPI (47% reduction after incubation for 72 hours at 37°C). We subsequently investigated the mechanism(s) that may underlie such inactivation. Oxidation of LDL is accompanied by the generation of various aldehydes, including malondialdehyde and 4-hydroxynonenal. Chemical modification with these aldehydes revealed a significant inverse correlation between the progressive loss of TFPI activity and both the increase in net electrical charge (r=−.93,P≤.0001) and the derivatization of free amino acid residues of LDL (r=−.90,P≤.0001). Specific chemical modification of lysine amino groups by acetylation similarly led to inactivation of LDL-associated TFPI activity. TFPI activity was almost totally abolished (<1.4%) when the TNBS reactivities of acetylated LDL, malondialdehyde-modified LDL, and 4-hydroxynonenal–modified LDL were 31%, 21%, and 43% that of native LDL, respectively. Our data demonstrate that expression of LDL-associated anticoagulant activity is markedly decreased as a consequence of the oxidative process, and suggest that the progressive aldehydic derivatization of apo B of LDL, and of the associated TFPI protein, may contribute to this phenomenon. Because tissue factor is overexpressed in the atheromatous plaque, it may exert a marked local procoagulant effect. The oxidative inactivation of LDL-associated TFPI will therefore effectively neutralize its inhibitory action on tissue factor activity, resulting in a disequilibrium in favor of coagulation.
T Lymphocytes Affect Smooth Muscle Cell Phenotype and Proliferation
Authors: Rolfe Barbara E. , Campbell Julie H. , Smith Nicole J. , Cheong Merron W. , Campbell Gordon R.
Published: June 2011

AbstractThe effects of rabbit T lymphocytes on rabbit aortic smooth muscle cell (SMC) phenotype and proliferation were investigated in vitro. SMCs seeded at confluent density in primary culture had a volume fraction of myofilaments (Vvmyo) of 49.8±2.6% after 3 days of culture, not significantly different from that of freshly dispersed cells (Vvmyo, 54.1±2.1%). Sister cultures of SMCs to which Concanavalin A–activated T lymphocytes or T lymphocyte–conditioned medium was added had significantly lower Vvmyo (35.5±2.2% and 31.6±2.3%, respectively) at the same time point. We have previously shown that a decrease in Vvmyo could be induced by the heparan sulfate–degrading activity of living macrophages and by commercial preparations of heparinase. While activated T lymphocytes also completely degraded heparan sulfate–rich35S-labeled extracellular matrix (an effect inhibited by the addition of 10 μg/mL heparin), no heparanase-like activity was detected in T lymphocyte–conditioned medium, indicating that for this cell type SMC phenotypic change is induced by a different mechanism. Incubation of the T lymphocyte–derived cytokine interferon gamma (IFN-γ) with freshly isolated rat SMCs caused a significant reduction in Vvmyo at day 2 in primary culture from 54.3±2.1% (control) to 35.4±3.0%. Furthermore, a neutralizing antibody specific for IFN-γ removed the effect of T lymphocytes and medium conditioned by them, thus positively identifying IFN-γ as the T lymphocyte factor responsible for this activity. T lymphocyte–conditioned medium was mitogenic for passaged (low Vvmyo) SMCs. Although SMC proliferation was inhibited by exogenous IFN-γ, two other T lymphocyte products, granulocyte-macrophage–colony stimulating factor and tumor necrosis factor–β, were found to stimulate proliferation, while interleukin-2 and interleukin-6 had no effect. It was concluded that T lymphocytes, by inducing SMC phenotypic change and stimulating proliferation, may play an important role in atherogenesis.
Fibrinogen and Silent Atherosclerosis in Subjects With Cardiovascular Risk Factors
Authors: Levenson Jaime , Giral Philippe , Razavian Mahmoud , Gariepy Jérôme , Simon Alain
Published: June 2011

Abstract
Fibrinogen may play an active role in the development and progression of atherosclerotic plaques. We assessed the association between fibrinogen levels and atherosclerotic plaques over three different arterial sites in an asymptomatic never-treated male population with increased cardiovascular risk. We included 652 men aged 40 to 60 years old with at least one of the following cardiovascular risk factors: cholesterol >6.2 mmol/L and/or systolic blood pressure ≥160 mm Hg and/or diastolic blood pressure ≥95 mm Hg, and/or because they smoked. Carotid and femoral arteries and the abdominal aorta were assessed by using ultrasonographic methods for the presence of plaque, and subjects were categorized according to the presence (or absence) and extent (one versus two or three sites) of plaque. Plasma fibrinogen was measured according to the thrombin-time method of Clauss. While the presence of atherosclerosis was significantly related to age, current smoking, systolic pressure, LDL cholesterol, and fibrinogen levels, the extent of atherosclerosis was related to age and triglyceride and fibrinogen levels. Multiple regression analysis indicated independent associations between fibrinogen and the presence and extent of atherosclerosis. Plaque prevalence was significantly more pronounced with increasing tertile of fibrinogen levels. The odds ratio of the upper to lower fibrinogen tertiles for the presence of plaque was 1.6 (95% confidence interval, 1.4 to 1.8) and 1.4 (95% confidence interval, 1.2 to 1.7) for its extent. Adjustment for other risk factors slightly reduced the association between fibrinogen and atherosclerosis. In conclusion, fibrinogen levels are related to atherosclerosis, supporting the hypothesis that increased fibrinogen may be one of the mechanisms linking cardiovascular risk factors to formation and progression of plaques.
Two New Immunogenetic Polymorphisms of the ApoB Gene and Their Effect on Serum Lipid Levels and Responses to Changes in Dietary Fat Intake
Authors: Ilmonen Marja , Heliö Tiina , Bütler René , Palotie Aarno , Pietinen Pirjo , Huttunen Jussi K. , Tikkanen Matti J.
Published: June 2011

AbstractIn previous studies, apoB polymorphisms have been shown to modify serum lipid responses to changes in dietary fat intake. The functionally important apoB DNA change or changes underlying these effects have, however, remained unknown. Using a single-strand conformation polymorphism analysis–based screening method, we identified two previously unreported apoB polymorphisms located close to each other in the 5′ region of apoB gene exon 26. This DNA segment corresponds to the binding site of monoclonal anti-apoB antibody D7.2. The two A→G changes at apoB cDNA nucleotides 5869 and 5896 produced an Asn→Ser change at amino acid 1887 and a His→Arg change at amino acid 1896. In the Finnish population, allele frequencies of the rare alleles of the apoB 1887 (Asn→Ser) and apoB 1896 (His→Arg) polymorphisms were .02 and .11, respectively. Both polymorphisms were shown to have an independent effect on the binding affinity of LDL with monoclonal antibody D7.2. The effect of these polymorphisms on serum lipid levels and responses to changes in dietary fat intake in 102 healthy free-living subjects was assessed. The apoB 1896 Arg allele was associated with a higher serum LDL cholesterol level during a low-fat, low-cholesterol diet in men.
Paracetamol Inhibits Copper Ion–Induced, Azo Compound–Initiated, and Mononuclear Cell–Mediated Oxidative Modification of LDL
Authors: Nenseter Marit S. , Halvorsen Bente , Rosvold Øyvind , Rustan Arild C. , Drevon Christian A.
Published: June 2011

Abstract
The effects of paracetamol and sodium salicylate on the susceptibility of LDL to oxidative modification were studied. LDL was subjected to Cu
2+
-, azo compound–, or peripheral blood mononuclear cell–initiated oxidation in the absence and presence of paracetamol and salicylate. Paracetamol (100 μmol/L; 25 μg LDL/mL) reduced the rate of formation of conjugated dienes and the amount of conjugated dienes formed during Cu
2+
-induced oxidation by 67% and 58%, respectively. Paracetamol (400 μmol/L; 100 μg LDL/mL) reduced the generation of lipid peroxides during Cu
2+
-induced oxidation by 43% (
P
<.05), the relative electrophoretic mobility in agarose gels by 16% (
P
<.05), and the amount of oxidized LDL taken up by J774 macrophages by 22% (
P
<.05). Paracetamol (100 μmol/L; 100 μg LDL/mL) reduced the 2,2′-azobis-(2-amidinopropane hydrochloride)–initiated lipid peroxidation by 70% (
P
<.05) and the relative electrophoretic mobility by 34% (
P
<.05). Paracetamol (100 μmol/L; 100 μg LDL/mL) reduced the amount of lipid peroxides generated in LDL during mononuclear cell–mediated oxidation by 69% (
P
<.01) and the relative electrophoretic mobility by 38% (
P
<.01). In comparison, 10 μmol/L α-tocopherol reduced the amount of lipid peroxides formed during cellular LDL oxidation and the relative electrophoretic mobility by 52% and 65%, respectively (
P
<.05). In the absence of paracetamol, SOD and catalase inhibited the modification of LDL (
P
<.05), suggesting that superoxide anions and hydrogen peroxide might be involved in the cell-mediated modification pathway. In the presence of paracetamol, SOD showed no additional inhibitory effect. The 1,1-diphenyl-2-pikrylhydracyl radical–scavenging test showed that paracetamol itself was a free-radical scavenger. In contrast, sodium salicylate (25 to 4000 μmol/L) showed no free radical–scavenging property and failed to protect LDL against mononuclear cell–mediated oxidation. In conclusion, the results indicate that paracetamol, but not salicylate, protects LDL against Cu
2+
-induced, azo compound–initiated, and mononuclear cell–mediated oxidative modification in vitro and that this may be due to the radical scavenger capacity of paracetamol.
Pravastatin Modulates Cholesteryl Ester Transfer From HDL to ApoB-Containing Lipoproteins and Lipoprotein Subspecies Profile in Familial Hypercholesterolemia
Authors: Guérin Maryse , Dolphin Peter J. , Talussot Corinne , Gardette Jean , Berthézène François , Chapman M. John
Published: June 2011

Abstract
Familial hypercholesterolemia (FH) results from genetic defects in the LDL receptor, and is characterized by a marked elevation in plasma LDL and by qualitative abnormalities in LDL particles. Because LDL particles are major acceptors of cholesteryl esters (CEs) from HDL, significant changes occur in the flux of CE through the reverse cholesterol pathway. To evaluate the effects of an HMG-CoA reductase inhibitor, pravastatin, on CE transfer from HDL to apo B–containing lipoproteins and on plasma lipoprotein subspecies profile in subjects with heterozygous FH, we investigated the transfer of HDL-CE to LDL subfractions and changes in both concentration and chemical composition of the apo B– and the apo AI–containing lipoproteins. After pravastatin treatment (40 mg/d) for a 12-week period, plasma LDL concentrations (mean±SD, 745.4±51.9 mg/dL) were reduced by 36% in patients with FH (n=6). By contrast, the qualitative features of the density profile of LDL subspecies in patients with FH, in whom the intermediate (
d
=1.029 to 1.039 g/mL) and dense (
d
=1.039 to 1.063 g/mL) subspecies were significantly increased relative to a control group, were not modified by pravastatin. In addition, no significant effect on the chemical composition of individual LDL subfractions was observed. Furthermore, plasma HDL concentrations were not modified, although the density distribution of HDL was normalized. Indeed, the HDL density peak was shifted towards the HDL
2
subfraction (ratios of HDL
2
to HDL
3
were 0.7 and 1.1 before and after treatment, respectively). Evaluation of plasma CE transfer protein (CETP) mass was performed with an exogenous CE transfer assay. Under these conditions, no modification of plasma CETP protein mass was induced by pravastatin administration. However, the rate of CE transfer from HDL to LDL was reduced by 24% by pravastatin (61±17 μg CE · h
−1
· mL
−1
plasma;
P
<.0005), although intermediate and dense LDL subfractions again accounted for the majority (71%) of the total CE transferred to LDL. Thus, pravastatin induced reduction of plasma CETP activity without change in the preferential targeting of the transfer of HDL-CE towards the denser LDL subfractions. In conclusion, pravastatin reduces the elevated flux of CE from HDL to apo B–containing lipoproteins in subjects with heterozygous FH as a result of a reduction in the LDL particle acceptor concentration.
Macrophage Uptake of Oxidized LDL Inhibits Lysosomal Sphingomyelinase, Thus Causing the Accumulation of Unesterified Cholesterol–Sphingomyelin–Rich Particles in the Lysosomes
Authors: Maor Irit , Mandel Hanna , Aviram Michael
Published: June 2011

AbstractMacrophage uptake of oxidatively modified LDL (Ox-LDL), unlike the uptake of acetylated LDL (Ac-LDL), resulted in lysosomal accumulation of unesterified cholesterol (UC). As sphingomyelin (SM) binds UC with high affinity, we considered whether lysosomes also accumulate Ox-LDL–derived SM, and if such a phenomenon could be involved in the lysosomal trapping of Ox-LDL–derived UC. Incubation of J-774 A.1 macrophages with Ox-LDL increased the lysosomal accumulations of UC by 75% and SM by 63% compared with the effect of Ac-LDL. The addition of chlorpromazine, an inhibitor of lysosomal sphingomyelinase (SMase), to macrophages that were incubated with [3H]cholesteryl ester–labeled Ac-LDL also led to lysosomal accumulation of both SM and UC. 7-Ketocholesterol (7-KC), the major oxysterol in Ox-LDL, inhibited lysosomal SMase in a cell-free system. The addition of 7-KC to cells in the presence of [3H]choline- or [3H]cholesteryl ester–labeled Ac-LDL led to macrophage accumulation of SM or UC, respectively. Niemann-Pick type C disease (NP-C) is an inherited cholesterol-storage disease in which lysosomal SMase activity is attenuated after uptake of LDL. Incubation of monocyte-derived macrophages from two NP-C patients with Ac-LDL or Ox-LDL resulted in an accumulation of UC in the lysosomes, whereas normal monocyte-derived macrophages accumulate UC in their lysosomes after incubation with Ox-LDL but not Ac-LDL. These results suggest that inhibition of lysosomal SMase in NP-C cells or by 7-KC is required for lysosomal accumulation of UC. Analysis of the macrophage lysosomal extract (following cell incubation with Ox-LDL) by density-gradient ultracentrifugation and gel-filtration chromatography revealed the presence of a particle consisting of UC, SM, 7-KC, and apoB-100. We conclude that 7-KC in Ox-LDL can inhibit lysosomal SMase, thus leading to the accumulation of SM, which binds UC avidly and inhibits its further cellular processing out of the lysosome. As UC-SM particles of lysosomal origin exist in the atherosclerotic lesion, the formation of such particles may result from an impaired processing of Ox-LDL by arterial wall macrophages during early atherogenesis.
Evidence for Nonesterified Fatty Acids as Modulators of Neutral Lipid Transfers in Normolipidemic Human Plasma
Authors: Lagrost Laurent , Florentin Emmanuel , Guyard-Dangremont Valérie , Athias Anne , Gandjini Hassan , Lallemant Christian , Gambert Philippe
Published: June 2011

Abstract
The relations between the level of plasma nonesterified fatty acid (NEFA) and both the mass concentration and activity of the cholesteryl ester transfer protein (CETP) were studied in fasted normolipidemic subjects. Plasma NEFA correlated positively with both CETP mass concentration (
r
=.50;
P
<.01) and the transfer of cholesteryl ester from HDL toward plasma VLDL+LDL (CET
HDL→VLDL+LDL
activity) (
r
=.46;
P
<.05) but not with the transfer of cholesteryl ester from LDL toward plasma HDL (CET
LDL→HDL
activity) (
r
=−.05; NS). The high binding capacity of albumin for NEFA was used to investigate whether lipoprotein-bound NEFAs were implicated in the modulation of the cholesteryl ester transfer reaction. As compared with nonsupplemented controls, the addition of an excess of fatty acid–free albumin (8 g/L) to total normolipidemic plasmas reduced CET
HDL→VLDL+LDL
activity (18.3±5.5% versus 9.8±3.1%;
P
<.0001) but not CET
LDL→HDL
activity (22.3±4.5% versus 23.3±5.1%; NS). Moreover, CET
HDL→VLDL+LDL
and CET
LDL→HDL
activities correlated negatively when measured in native plasma (
r
=−.45;
P
<.05) but positively when measured in albumin-supplemented plasma (
r
=.40;
P
<.05). In long-term incubation experiments, lipoprotein-bound NEFA increased the net mass transfer of cholesteryl esters from HDL toward VLDL+LDL but reduced the net mass transfer of triglycerides in the opposite direction, from VLDL+LDL toward HDL. Taken together, data of the present study brought strong and concordant arguments in favor of a dual effect of plasma NEFA in modulating both the mass and the activity of CETP in vivo.
Effect of Dietary Cholesterol and Fat on the Expression of Hepatic Sterol 27-Hydroxylase and Other Hepatic Cholesterol-Responsive Genes in Baboons (
Papio
Species)
Authors: Kushwaha Rampratap S. , Guntupalli Bharathi , Rice Karen S. , Carey K. Dee , McGill Henry C.
Published: June 2011

Abstract
Our studies of baboons with low and high responses to dietary cholesterol and fat suggest that low-responding baboons increase the activity of hepatic sterol 27-hydroxylase, an important enzyme of bile acid synthesis, considerably more than do high-responding baboons when challenged with a high-cholesterol, high-fat (HCHF) diet. The present studies were conducted to determine whether hepatic sterol 27-hydroxylase mRNA levels and plasma 27-hydroxycholesterol concentrations also differed with dietary responsiveness. Sixteen adult male baboons with a wide range of VLDL cholesterol plus LDL cholesterol (VLDL+LDL cholesterol) response to an HCHF diet were selected. They were examined first while on a chow diet and then after 1, 3, 6, 10, 18, 26, 36, 52, 72, and 104 weeks on the HCHF diet. Plasma and lipoprotein cholesterol concentrations increased rapidly during the first 3 weeks and stabilized thereafter. On the basis of the response in VLDL+LDL cholesterol, we selected five low-responding, four medium-responding, and five high-responding baboons for more intensive study in more detail. In low responders, the major increase in serum cholesterol concentration was in HDL cholesterol, whereas in medium and high responders it was in both VLDL+LDL and HDL cholesterol. In low and medium responders, serum or VLDL+LDL cholesterol did not change after 3 weeks of consumption of the HCHF diet, whereas in high responders VLDL+LDL cholesterol declined between 78 and 104 weeks. In low and medium responders, plasma 27-hydroxycholesterol concentrations and hepatic sterol 27-hydroxylase mRNA levels increased rapidly during the first 10 weeks, declined thereafter, and stabilized at 26 weeks in low responders and at 40 weeks in medium responders. In high responders, plasma 27-hydroxycholesterol concentration did not increase. The increases in hepatic sterol 27-hydroxylase mRNA and plasma 27-hydroxycholesterol in medium responders were intermediate between those in high- and low-responding baboons. The expression of hepatic mRNA for other cholesterol-responsive genes did not differ between high- and low-responding baboons. Plasma LDL cholesterol concentrations were negatively correlated with plasma 27-hydroxycholesterol concentrations and hepatic sterol 27-hydroxylase mRNA levels when the animals had been on the HCHF diet for 10 weeks. These studies suggest that hepatic sterol 27-hydroxylase may be an important regulator of responsiveness to dietary cholesterol and fat in baboons.
Cholesterol Efflux, Cholesterol Esterification, and Cholesteryl Ester Transfer by LpA-I and LpA-I/A-II in Native Plasma
Authors: Huang Yadong , von Eckardstein Arnold , Wu Shili , Assmann Gerd
Published: June 2011

Abstract
HDLs encompass structurally heterogeneous particles that fulfill specific functions in reverse cholesterol transport. Two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) of normal plasma and subsequent immunoblotting with anti–apolipoprotein (apo) A-I antibodies differentiates an abundant particle with electrophoretic α-mobility and less abundant particles with electrophoretic pre-β-mobility (preβ
1
–LpA-I, preβ
2
–LpA-I, preβ
3
–LpA-I). Immunodetection with anti–apoA-II antibodies identifies a single particle with α-mobility. To differentiate α-migrating HDL without apo A-II (α–LpA-I) from those with apoA-II (α–LpA-I/A-II), we combined 2D-PAGGE with immunoadsorption of apoA-II. Incubation of plasma with [
3
H]cholesterol-labeled fibroblasts in combination with immunosubtracting 2D-PAGGE allowed us to analyze the role of α–LpA-I and α–LpA-I/A-II in the uptake and esterification of cell-derived cholesterol in native plasma. Depending on the duration of incubations with cells, α-LpA-I took up two to four times more [
3
H]cholesterol than α–LpA-I/A-II. Irrespective of the duration of incubation, two to three times more [
3
H]cholesteryl esters accumulated in α–LpA-I than in α–LpA-I/A-II. Subsequent incubations in the presence of an inhibitor of lecithin:cholesterol acyltransferase led to preferential accumulation of [
3
H]cholesteryl esters in α–LpA-I/A-II. In conclusion, our data indicate that α–LpA-I is more effective than α–LpA-I/A-II in both uptake and esterification of cell-derived cholesterol. Moreover, α–LpA-I/A-II appears to accumulate cholesteryl esters, at least partially, from α–LpA-I.
Decrease in Scavenger Receptor Expression in Human Monocyte–Derived Macrophages Treated With Granulocyte Macrophage Colony-Stimulating Factor
Authors: van der Kooij Maaike A. , Morand Olivier H. , Kempen Herman J. , van Berkel Theo J.C.
Published: June 2011

AbstractTo determine whether scavenger receptors are susceptible to regulation by granulocyte macrophage colony-stimulating factor (GM-CSF), a macrophage-specific cytokine, human monocytes were differentiated into macrophages in the absence or presence of 20 U/mL GM-CSF. Binding, uptake, and degradation of acetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL) were measured. Treatment with GM-CSF resulted in a significant twofold to threefold decrease in the number of binding sites for Ac-LDL and Ox-LDL on the surface of macrophages without affecting the affinity of the receptor for these ligands. Competition experiments revealed that two binding sites were responsible for the recognition and uptake of Ac-LDL: one specific for Ac-LDL and one that recognized both Ac-LDL and Ox-LDL. No binding site specific for Ox-LDL could be detected in either control or GM-CSF–treated macrophages. Treatment of human monocyte–derived macrophages with GM-CSF resulted in a decrease of the Ac-LDL/Ox-LDL receptor but did not affect the binding site specific for Ac-LDL. Northern blot analysis showed that mRNA levels of both types I and II scavenger receptor were reduced in macrophages differentiated in the presence of GM-CSF. Human macrophages that were differentiated in the presence of GM-CSF accumulated ≈50% fewer cholesteryl esters. Taken together, these results indicate that GM-CSF can downregulate both types I and II scavenger receptor in human monocyte–derived macrophages, which might have implications for foam cell formation.
Lp(a) Levels and Atherosclerotic Vascular Disease in a Sample of Patients With Familial Hypercholesterolemia Sharing the Same Gene Defect
Authors: Carmena Rafael , Lussier-Cacan Suzanne , Roy Madeleine , Minnich Anne , Lingenhel Arno , Kronenberg Florian , Davignon Jean
Published: June 2011

AbstractThere is considerable variation in the severity of cardiovascular disease among patients with familial hypercholesterolemia (FH). Some reports have suggested that plasma lipoprotein(a) [Lp(a)] levels may explain such variation and that FH subjects deficient in LDL receptors, especially those with coronary heart disease, tend to have elevated Lp(a) levels. We have investigated the possible role of the LDL receptor in determining plasma Lp(a) levels in a genetically homogeneous FH population and the contribution of Lp(a) to cardiovascular risk. A total of 98 FH subjects and 66 healthy first- and second-degree relatives from 30 families with FH due to the French-Canadian >10-kilobase deletion of the LDL receptor gene were studied. A reference group of 392 normolipidemic French-Canadian participants in a Heart Health Survey was used for comparison. FH subjects were subdivided into subsets of 63 individuals free from atherosclerotic vascular disease (AVD) and 35 individuals with AVD. A complete cardiovascular evaluation was performed, and plasma lipid, lipoprotein, and Lp(a) levels were measured in all subjects in the absence of medication. Apolipoprotein (a) [apo(a)] phenotype was determined in 112 of FH and non-FH subjects. The log-transformed values for plasma Lp(a) were not significantly different among the three groups: 0.98±0.54 (mean±SD) in FH subjects with AVD, 0.89±0.51 in FH subjects without AVD, and 0.82±0.64 in their relatives. The distribution of the apo(a) phenotypes did not differ between the FH and non-FH groups. Comparison of two age- and sex-matched subgroups of FH subjects, with and without AVD, failed to show any differences in Lp(a) level. However, mean Lp(a) log values in the reference group (n=392) were significantly lower than values obtained for the total FH group (0.79±0.57 versus 0.92±0.52, respectively;P<.05) but were not different from those of the unaffected family members. Thus, in our sample, the LDL receptor appears not to influence plasma Lp(a) levels; rather, these levels reflect shared apo(a) genes. The cardiovascular risk in this group of subjects with FH was related to age, male sex, total and LDL cholesterol, and higher apoB but not Lp(a) levels.
Sex Differences in Coagulation and Fibrinolysis in White Subjects With Non–Insulin-Dependent Diabetes Mellitus
Authors: Mansfield Michael W. , Heywood Daniella M. , Grant Peter J.
Published: June 2011

Abstract
The increase in cardiovascular risk associated with having non–insulin-dependent diabetes mellitus (NIDDM) is far greater in women than men. Conventional risk factors do not account for this excess, and attention has focused on the possible contribution of abnormalities of fibrinolysis and coagulation in NIDDM. In the general population a number of hemostatic factors have been shown to predict the occurrence or progression of coronary artery disease. To investigate sex differences in coagulation and fibrinolysis in NIDDM, we measured levels of fibrinogen, factor VII:C, von Willebrand factor, plasminogen activator inhibitor-1, and tissue plasminogen activator in 213 NIDDM subjects (124 men and 89 women) who were not receiving insulin therapy. The women had higher levels of factor VII:C (144% versus 120.5% in men,
P
<.0005) and plasminogen activator inhibitor-1 activity (25.6 versus 17.0 U/mL), and these differences remained significant when account was taken of the higher body mass index (29.6 versus 28.0 kg/m
2
,
P
=.02), glycosylated hemoglobin (7.2% versus 6.8%,
P
<.05), and cholesterol levels (6.3 versus 5.7 mmol/L,
P
<.0005) in women than men. In contrast, levels of fibrinogen (3.2 versus 3.1 g/L), tissue plasminogen activator antigen (10.6 versus 11.2 ng/mL), and von Willebrand factor (1.27 versus 1.23 IU/mL) were no different between women and men, respectively. These results suggest that elevated levels of plasminogen activator inhibitor-1 and factor VII:C may contribute to the increased cardiovascular risk of NIDDM that is particularly marked in women.
A Method for the Assessment of Hypoxia in the Arterial Wall, With Potential Application In Vivo
Authors: Björnheden T. , Evaldsson M. , Wiklund O.
Published: June 2011

Abstract
According to the anoxemia theory of atherosclerosis, an imbalance between the demand for and supply of oxygen in the arterial wall is a key factor in the development of atherosclerotic lesions. Direct in vitro and in situ measurements have shown that P
o
2
is decreased in the inner part of the media, but the degree of hypoxia in vivo or the distribution of hypoxia along the arterial tree is not known. We applied a hypoxia marker, 7-(4′-(2-nitroimidazol-1-yl)-butyl)-theophylline (NITP), to develop a method for the detection of hypoxia in the arterial wall. Immunoperoxidase and immunofluorescence were used to detect the marker, and a clearly P
o
2
-dependent staining was observed in the media of rabbit and swine aorta in vitro. The cutoff P
o
2
level was probably around 2 to 3 mm Hg. In experimental atherosclerotic lesions in the rabbit the marker seemed to bind to foam cells that were already at a higher surrounding P
o
2
, which might reflect a higher local oxygen consumption. The binding of the marker to endothelial cells was not P
o
2
dependent. One explanation for this finding could be that the marker was metabolized via a non–oxygen-dependent pathway in these cells. We propose that this method may be used to assess arterial wall hypoxia in vivo. Furthermore, the spatial resolution allows the detection of local variations within the arterial tree.
Apoptosis of Vascular Smooth Muscle Cells Induced by In Vitro Stimulation With Interferon-γ, Tumor Necrosis Factor–α, and Interleukin-1β
Authors: Geng Yong-Jian , Wu Qi , Muszynski Maria , Hansson Göran K. , Libby Peter
Published: June 2011

Abstract
Recent studies have documented evidence for the death of smooth muscle cells (SMCs) within advanced human atheroma. These lesions contain macrophages and T lymphocytes in addition to SMCs. We therefore investigated whether interferon-γ (IFN-γ), a cytokine secreted by T lymphocytes, or interleukin-1β (IL-1β) and tumor necrosis factor–α (TNF-α), two cytokines characteristically produced by activated macrophages, can trigger apoptosis of vascular SMCs. Simultaneous treatment with IFN-γ and TNF-α and/or IL-1β but not with each cytokine alone promoted death of human and rat SMCs. Exposure for 48 hours to a combination of IFN-γ (400 U/mL), TNF-α (400 U/mL), and IL-1β (100 U/mL) significantly (
P
<.001) increased the accumulation of oligonucleosomes comprising DNA fragments and histones in human SMCs. Electrophoresis of genomic DNA showed internucleosomal fragments of genomic DNA isolated from the cytokine-cotreated SMCs of both humans and rats. These cells exhibited morphological changes typical of apoptosis, including cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. In situ 3′ end labeling of DNA fragments with terminal transferase confirmed the fragmentation of genomic DNA in these cells. Simultaneous treatment with IFN-γ and TNF-α or IL-1β induced elaboration of nitrite, an end product of nitric oxide, in rat but not human SMCs.
N
G
-monomethyl-
l-
arginine inhibited nitrite accumulation and also partly blocked cytokine-induced apoptosis of rat SMCs but had little effect on human SMCs, suggesting operation of both nitric oxide–dependent and –independent mechanisms for cytokine-induced apoptosis in vascular SMCs. Production of immune cytokines by vascular cells and/or infiltrating leukocytes may regulate apoptotic death of SMCs during atherogenesis.
Time Course of Cellular Proliferation, Intimal Hyperplasia, and Remodeling Following Angioplasty in Monkeys With Established Atherosclerosis
Authors: Geary Randolph L. , Williams J. Koudy , Golden Deborah , Brown Deanna G. , Benjamin Marshall E. , Adams Michael R.
Published: June 2011

Abstract
Animal models of arterial injury have failed to predict effective therapy to prevent restenosis in humans. While this may relate to species differences in the control of smooth muscle cell growth, many studies have used nonatherosclerotic animals, thereby failing to consider the importance of atherosclerosis in the response to injury. In an attempt to model human restenosis more accurately, we characterized the response to angioplasty in atherosclerotic monkeys. Twenty-one cynomolgus monkeys were fed an atherogenic diet for 36 months (plasma cholesterol, 12±1 mmol/L [470±23 mg/dL]). Angioplasty was then performed in the left iliac artery. After 4, 7, 14, or 28 days, bromodeoxyuridine was given to label proliferating cells, and iliac arteries were fixed in situ at physiological pressure (5 or 6 animals at each time point). Comparisons were made between injured and uninjured iliac arteries within each animal. Angioplasty often fractured the intimal plaque and media, transiently increasing lumen caliber (4 days: lumen area, 232.5±80.3% of control) and artery size as reflected by external elastic lamina area (EEL). EEL and lumen caliber returned to baseline by 7 days. Proliferation was increased throughout the artery wall at 4 and 7 days and later declined to control rates (4 days, injured versus uninjured: adventitia, 45.0±6.2% versus 16.3±7.2%; media, 8.6±2.6% versus 0.6±0.1%; intima, 16.0±5.6% versus 7.8±3.1%). The intima thickened markedly from 14 to 28 days, but an increase in EEL generally prevented further loss of the short-term gain in lumen caliber (28 days, percent of control: intimal area, 342.8±88.9%; EEL area, 150.2±28.9%; lumen area, 119.3±21.3%). The response to angioplasty in atherosclerotic monkeys appears to closely resemble that in humans. Plaque fracture, delayed recoil, intimal hyperplasia, and remodeling may each be important in determining late lumen caliber. This primate model should prove valuable in defining cellular and biochemical mediators of human restenosis.
Platelet Activation in Acute Myocardial Infarction and Unstable Angina Is Inhibited by Nitric Oxide Donors
Authors: Langford E.J. , Wainwright R.J. , Martin J.F.
Published: June 2011

Abstract
Platelet activation and thrombus formation within the coronary artery are major factors in acute myocardial infarction (AMI) and unstable angina (UA), and continuing platelet activation is associated with an adverse prognosis. We assessed platelet activation by using flow cytometry to measure platelet surface expression of P-selectin and glycoprotein IIb/IIIa in 20 patients with AMI and 20 with UA, all of whom were treated with aspirin. Platelet studies were repeated after the infusion of a nitric oxide donor (glyceryl trinitrate or
S
-nitrosoglutathione) that produced a fall in mean arterial pressure of no more than 10 mm Hg. P-selectin was expressed on 2.5% (range, 1.4% to 6.3%) of platelets from AMI and 2.3% (range, 1.6% to 3.3%) from UA subjects compared with 1.0% (range, 0.6% to 1.9%) of platelets from 20 control volunteers without angina (
P
<.001). Glycoprotein IIb/IIIa expression was 101.6±2.7 arbitrary units of relative fluorescence in AMI and 100.2±3.3 in UA compared with 87.8±2.5 in control subjects (
P
<.01). In both AMI and UA,
S-
nitrosoglutathione reduced P-selectin (
P
<.001) and glycoprotein IIb/IIIa (
P
<.05) expression, as did glyceryl trinitrate (
P
<.02 and
P
<.01, respectively). In 3 of 20 patients receiving glyceryl trinitrate the lowest dose was not tolerated due to headache or hypotension. These findings show that platelet activation persists in AMI and UA despite aspirin treatment and that this can be inhibited by using glyceryl trinitrate or
S-
nitrosoglutathione.
S-
nitrosoglutathione is better tolerated at the doses required.
Adhesion of Blood Platelets Is Inhibited by VCL, a Recombinant Fragment (Leucine
504
to Lysine
728
) of von Willebrand Factor
Authors: Sixma Jan J. , IJsseldijk Martin J.W. , Hindriks George , van Zanten G. Henrita , Gorecki Marian , Panet Amos , Garfinkel Leonard I. , de Groot Philip G.
Published: June 2011

Abstract
VCL, fragment Leu
504
to Lys
728
of von Willebrand factor (vWF) expressed in
Escherichia coli
, contains the glycoprotein (GP) Ib–binding domain of vWF. This fragment inhibited ristocetin-induced platelet aggregation with an IC
50
of 0.2 μmol/L and botrocetin-induced aggregation with an IC
50
of 0.08 μmol/L. We studied the antiadhesive profile of VCL by adding it to blood that was circulated over various adhesive surfaces. VCL inhibited adhesion to endothelial cell matrix, which served as a model of the vessel wall. Maximal inhibition at a high shear rate of 1600 s
−1
was stronger (60%) than at a low shear rate of 300 s
−1
(40%). Half maximal inhibition was found to be 1.5 μmol/L at both shear rates. The role of various adhesive molecules was investigated in more detail by coating glass coverslips with collagen type I, laminin, fibronectin, or vWF. Fibrinogen was studied as well. Platelet adhesion to laminin and vWF was not inhibited by VCL. Adhesion to collagen, fibronectin, and fibrinogen was particularly inhibited at a high shear rate. VCL coated to a coverslip caused a concentration-dependent adhesion that was blocked by antibodies against GPIb, which block interaction with vWF. Binding studies showed a nonsaturable ristocetin binding of VCL to platelets that was blocked by vWF or inhibitory antibodies against GPIb. Binding to collagen was weak, and VCL did not inhibit binding of vWF at a 5000-fold excess. From these data, we conclude that VCL inhibits adhesion in all cases in which adhesion is vWF dependent by competing for vWF binding to activated GPIb. The lack of inhibition of adhesion to vWF as a single molecule may be explained by assuming that this adhesion is determined by interaction of nonactivated GPIb with vWF that has been changed in conformation by adsorption. Studies investigating thrombus formation on the connective tissue of an atherosclerotic plaque in a human coronary artery showed that VCL was able to partially prevent this thrombus formation. VCL may be of value in preventing adhesion and thrombus formation under conditions in which these processes are dependent on vWF.
High Concentrations of Active Plasminogen Activator Inhibitor-1 in Porcine Coronary Artery Thrombi
Authors: Fay William P. , Murphy Joseph G. , Owen Whyte G.
Published: June 2011

Addition of exogenous plasminogen activator inhibitor-1 (PAI-1) to fibrin clots inhibits fibrinolysis in vivo. However, it is unknown whether the localized concentrations of active PAI-1 necessary to produce this antifibrinolytic effect can be recruited to acute arterial thrombi by endogenous mechanisms. We measured PAI-1 activity and antigen in porcine coronary artery thrombi that formed in response to acute vascular injury. Mean PAI-1 activity in thrombi (n=5) was 36±5.1 μg/mL, which is >2000 times its concentration in normal porcine plasma. The presence of markedly elevated concentrations of active PAI-1 in thrombi was confirmed by an immunoactivity assay and by demonstrating formation of sodium dodecyl sulfate–stable complexes after addition of
125
I-urokinase to thrombus extracts. Comparative analysis of PAI-1 antigen by Western blotting and urokinase inhibition assay suggested that approximately one third of thrombus-associated PAI-1 was active. Histological examination of coronary thrombi revealed that they consisted predominantly of dense aggregates of platelets with interspersed islands of fibrin, which closely resemble the histological appearance of thrombi in patients with myocardial infarction and unstable angina pectoris. Washed porcine platelets prepared from peripheral blood contained sufficient PAI-1 antigen and activity to account for the concentrations observed in coronary artery thrombi. However, the specific activity of human platelet PAI-1 was lower than that of porcine platelet PAI-1 (2% versus 50% active, respectively), and human platelets inhibited in vitro fibrinolysis to a lesser extent than did porcine platelets. These results indicate that active PAI-1 accumulates in porcine coronary artery thrombi in concentrations markedly higher than those present in plasma and that PAI-1 may be an important determinant of the known resistance of platelet-rich thrombi to lysis by tissue-type plasminogen activator. These studies also underscore the importance of considering possible species differences in protein function when comparing animal models of thrombosis to acute coronary thrombosis in humans.
Sustained Inhibition of Whole-Blood Clot Procoagulant Activity by Inhibition of Thrombus-Associated Factor Xa
Authors: McKenzie Clark R. , Abendschein Dana R. , Eisenberg Paul R.
Published: June 2011

Progression of arterial thrombosis partly depends on thrombus-associated thrombin and activated factor X (Xa) activity. However, whether Xa or thrombin is the most appropriate target for inhibition of recurrent thrombosis is unknown. This study was designed to determine whether inhibition of Xa results in more sustained attenuation of thrombus-associated procoagulant activity than does inhibition of thrombin. Clots prepared ex vivo from human whole blood and pathological arterial thrombi from patients were preincubated in citrated plasma containing no inhibitor, 0.5 to 1 U/mL heparin, 0.5 to 1 μmol/L hirudin, 5 to 10 μmol/L tick anticoagulant peptide (TAP), 0.15 to 3 μmol/L tissue factor pathway inhibitor (TFPI), or a combination of 1 μmol/L hirudin and 10 μmol/L TAP for 2 hours. After preincubation the clots were removed from first-stage plasma, extensively washed in phosphate-buffered saline, and added to nonanticoagulated whole blood. Clots preincubated in plasma without inhibitors induced marked activation of the coagulation system in whole blood, as characterized by greater increases in the concentration of fibrinopeptide A (FPA) over 7 minutes than in blood without added clots (1522±568 compared with 117±170 ng/mL,
P
<.01). Preincubation of clots with heparin or hirudin did not attenuate the increases in FPA in whole blood. In contrast, compared with incubation without an inhibitor, preincubation of clots with TAP or TFPI markedly attenuated the increases in FPA when clots were added to whole blood (551±316 and 508±208 ng/mL, respectively,
P
<.01). Similar results were obtained with arterial thrombi from patients. Inhibition of Xa but not of thrombin results in sustained attenuation of thrombus-associated procoagulant activity. Uninhibited thrombus-associated Xa activity may account for the increases in thrombin activity that are commonly observed in clinical trials after discontinuation of thrombin inhibitors.
Postprandial Elevation of Activated Factor VII in Young Adults
Authors: Kapur Rajesh , Hoffman Carol J. , Bhushan Vikas , Hultin Mae B.
Published: June 2011

The Northwick Park Heart Study found that factor VII (FVII) activity was a risk factor for ischemic heart disease, and other studies based on indirect assays of activated factor VII (FVIIa) found an elevation of FVIIa postprandially. We hypothesized that postprandial elevation of FVIIa would produce intermittent activation of factor X to Xa and, subsequently, prothrombin to thrombin. We chose to study postprandial activation of coagulation with a new assay specific for FVIIa that uses soluble tissue factor and with a prothrombin fragment 1+2 (F
1+2
) assay to detect the activation of prothrombin by factor Xa. We fed a high-fat breakfast (30 g/m
2
) to 30 healthy volunteer subjects (30.8±9.8 years; range, 20 to 49 years) on no medication. Fasting blood samples were collected for FVIIa, FVII antigen (FVIIag), and F
1+2
as well as triglycerides and total and HDL cholesterol. A significant difference was found between fasting (2.82±1.49 ng/mL, mean±SD) and 6-hour postprandial (3.45±2.08 ng/mL) FVIIa levels (
P
<.004); FVIIag did not change significantly (mean, 0.89 U/mL fasting and 0.90 U/mL at 6 hours). In contrast, F
1+2
levels were slightly lower 6 hours postprandially than fasting (median, 0.39 versus 0.44 nmol/L,
P
<.02). Four-hour postprandial triglyceride levels correlated significantly (ρ=0.51,
P
<.02) with 6-hour postprandial FVIIag but not with 6-hour postprandial FVIIa. Postprandial F
1+2
levels (at 6 hours) correlated significantly (ρ=0.39,
P
<.04) with fasting FVIIag levels but not with 6-hour postprandial FVIIa levels. Thus, the basal FVIIag level, in the fasting state, may be involved in control of the generation of F
1+2
. We found a postprandial increase in FVIIa levels after a dietary fat load but did not find a concomitant postprandial burst of activation of factor X and prothrombin as measured by F
1+2
. Further studies are to test whether postprandial FVIIa generation leads to enhanced activation of coagulation.