bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Effects of the Acyl-Coenzyme A:Cholesterol Acyltransferase Inhibitor 58-035 on Foam Cell Development in Primary Human Monocyte–Derived Macrophages
Authors: Rodriguez Annabelle , Bachorik Paul S. , Wee Siok-Bi
Published: June 2011

Abstract
—We examined the effect of acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors on intracellular cholesterol stores in primary human monocyte–derived macrophages (HMMs) during foam cell formation. HMMs were exposed to acetylated low density lipoprotein (acLDL, 500 μg protein per mL) with or without 58-035 (1 to 10 μg/mL) or CI-976 (2 μg/mL) for 2 to 48 hours. Total cholesterol (TC) and esterified cholesterol (EC) mass was significantly lower while unesterified cholesterol (UC) increased slightly in cells incubated with acLDL plus ACAT inhibitors. Sterol mass was also measured in cells coincubated with acLDL (500 μg protein per mL) with or without 58-035 (2 μg/mL), high density lipoprotein (HDL, 400 μg protein per mL), or HDL+58-035 for 48 hours. TC and EC were 23% and 55% lower, respectively (
P
<0.0004), while UC was 11% higher (
P
<0.04) in cells incubated with acLDL plus 58-035. In contrast, coincubation with HDL alone did not significantly affect TC, EC, or UC mass compared with acLDL alone. The effect of 58-035 could not be explained by cytotoxicity, because adenine release, secreted lactate dehydrogenase, glucose utilization, and cell protein were similar in cells exposed to acLDL regardless of the presence of 58-035. We investigated several potential mechanisms for the decreased TC mass, including increased UC efflux and decreased acLDL binding and uptake. Efflux was measured in cells exposed to [1,2-
3
H]cholesteryl oleate–labeled acLDL, unlabeled control acLDL, and native untreated acLDL (500 μg protein per mL) with or without 58-035 (5 μg/mL) for 24 or 48 hours. UC efflux increased in a time-dependent manner from cells exposed to acLDL plus 58-035 compared with cells exposed to acLDL alone (
P
<0.04). High-affinity binding was measured in cells exposed to
125
I-acLDL (5 μg protein per mL) with or without excess unlabeled acLDL (100 or 500 μg protein per mL) for 4 hours at 4°C. Specific acLDL binding, uptake, and total degradation were significantly lower when 58-035 was present during cholesterol enrichment compared with cells exposed to acLDL alone (
P
<0.001). Unlike the effects of ACAT inhibitors on foam cell formation in rodent macrophages, these compounds lowered TC accumulation in HMMs during foam cell formation by limiting the uptake of acLDL and enhancing UC efflux. They may offer promise as drug therapies for atherosclerosis.
Human Serum Paraoxonase/Arylesterase’s Retained Hydrophobic
N
-Terminal Leader Sequence Associates With HDLs by Binding Phospholipids
Authors: Sorenson Robert C. , Bisgaier Charles L. , Aviram Michael , Hsu Cary , Billecke Scott , La Du Bert N.
Published: June 2011

Abstract
—In serum, human paraoxonase/arylesterase (PON1) is found exclusively associated with high density lipoprotein (HDL) and contributes to its antiatherogenic properties by inhibiting low density lipoprotein (LDL) oxidation. Difficulties in purifying PON1 from apolipoprotein A-I (apoA-I) suggested that PON1’s association with HDL may occur through a direct binding between these 2 proteins. An unusual property of PON1 is that the mature protein retains its hydrophobic
N
-terminal signal sequence. By expressing in vitro a mutant PON1 with a cleavable
N
-terminus, we demonstrate that PON1 associates with lipoproteins through its
N
-terminus by binding phospholipids directly rather than binding apoA-I. Nonetheless, apoA-I stabilized arylesterase activity more than did phospholipid alone, apoA-II, or apoE. Consequently, we studied the role of apoA-I in PON1 expression and HDL association in mice genetically deficient in apoA-I. Though present in HDL fractions at decreased levels, PON1 arylesterase activity was less stable than in control mice. Furthermore, PON1 could be competitively removed from HDL by phospholipids, suggesting that PON1’s retained
N
-terminal peptide allows transfer of the enzyme between phospholipid surfaces. Thus, our data suggest that PON1 is stabilized by apoA-I, and its binding to HDL and physiological distribution are dependent on the direct binding of the retained hydrophobic
N
-terminus to phospholipids optimally presented in association with apoA-I.
Cystatin C as a Determinant of Fasting Plasma Total Homocysteine Levels in Coronary Artery Disease Patients With Normal Serum Creatinine
Authors: Bostom Andrew G. , Bausserman Linda , Jacques Paul F. , Liaugaudas Gintaras , Selhub Jacob , Rosenberg Irwin H.
Published: June 2011

Abstract
—Serum creatinine, a surrogate for both renal function and homocysteine generation, is a determinant of fasting plasma total homocysteine levels in coronary artery disease (CAD) patients. We hypothesized that among stable-CAD patients with normal creatinine levels (ie, ≤1.4 mg/dL), serum cystatin C, a more sensitive indicator of glomerular filtration rate, would better predict fasting total homocysteine levels in comparison with serum creatinine. Fasting plasma total homocysteine, folate, vitamin B
12
, and pyridoxal 5′-phosphate levels, along with serum cystatin C, creatinine, and albumin levels, were determined in 164 consecutive stable-CAD patients (mean±SD age, 61±9 years; 78.7% men) whose serum creatinine level was ≤1.4 mg/dL. All subjects were examined at least 3 to 4 months after the widespread availability of cereal grain flour products fortified with folic acid. General linear modeling with ANCOVA revealed that serum cystatin C (
P
<0.001), B
12
(
P
<0.001), age (
P
=0.002), albumin (
P
=0.008), and sex (
P
=0.024) were independent determinants of fasting total homocysteine levels. Cystatin C alone determined over half of the variability (ie,
R
2
) in total homocysteine levels accounted for by these 5 independent regressors. In contrast, creatinine, folate, and pyridoxal 5′-phosphate were not independently predictive of fasting total homocysteine levels (
P
>0.2). Consistent with the impact of folic acid fortification of cereal grain flour in the general population, only 1 of the CAD subjects (0.6%) had a plasma folate level <3 ng/mL. We conclude that serum cystatin C levels may reflect subtle decreases in renal function that independently predict fasting total homocysteine levels among stable-CAD patients with normal serum creatinine.
Kininogens Are Antithrombotic Proteins In Vivo
Authors: Colman Robert W. , White John V. , Scovell Sherry , Stadnicki Antoni , Sartor R. Balfour
Published: June 2011

Abstract
—Kininogens have recently been shown to possess antiadhesive, anticoagulant, and profibrinolytic properties and can inhibit platelet activation at low thrombin concentrations. To test whether kininogens have antithrombotic properties in vivo, we devised a model of limited arterial injury confined to removal of the endothelium. Brown-Norway Katholiek strain rats with an absence of low- and high-molecular-weight kininogen due to a single point mutation, A163T, were compared in the thrombosis model to the wild-type animals, which were otherwise genetically identical. Despite an equivalent vascular injury, the mean time (±SEM) for a 90% decrease in flow measured by laser Doppler was 38.4±17 minutes in the kininogen-deficient rats compared with 194±29 minutes in the wild-type animals (
P
<0.002). The degree of vascular injury was the same. No evidence for disseminated intravascular coagulation (decrease in factor V, antithrombin, or fibrinogen) or excessive fibrinolysis (elevation of fibrinogen degradation products) was found in either group of animals. The results suggest that kininogens have antithrombotic properties at low concentrations of thrombin and that inhibitory peptides derived from kininogen may constitute a new antithrombotic strategy.
Dietary lipids, sugar, fiber and mortality from coronary heart disease. Bivariate analysis of international data.
Authors: Liu K , Stamler J , Trevisan M , Moss D
Published: June 2011

Univariate analyses with data for the years 1954-1969 from 20 economically advanced countries showed that a combined dietary lipid score based on per capita consumption of saturated fat, cholesterol, and polyunsaturated fat (equation of Keys, et al.) and the consumption of refined and processed sugars were both highly correlated with age-standardized coronary heart disease (CHD) mortality rates for the years 1969-1973 for both men and women aged 35-74 years (r values of 0.54 to 0.72). Fiber intake, estimated as the sum of calories available from vegetables, fruits, grains, and legumes, yielded a significant inverse correlation with CHD mortality rates (r = -0.49 to -0.68). These three dietary variables are highly intercorrelated. With bivariate analyses (2 X 2 cross-classification and analysis of variance), lipid score, but neither that of sucrose or fiber, was consistently and significantly related to CHD mortality. However, with both higher and lower lipid scores, CHD mortality rates tended to be slightly (nonsignificantly) higher with higher sugar values and with lower fiber values.
Atherosclerosis in rabbits fed a low cholesterol diet for five years.
Authors: Wilson R B , Miller R A , Middleton C C , Kinden D
Published: June 2011

Rabbits were fed a low cholesterol atherogenic diet for up to 5 years. Arterial lesions during the first 12 months consisted of smooth muscle cell and lipid accumulation in the intima, with smaller amounts of elastin and collagen. By 24 months, considerable degeneration and necrosis of smooth muscle foam cells had occurred, lipid had decreased in relative proportion, and collagen was predominant. These trends continued during the final 3 years. By 48 months most plaques were calcified and a few had hemorrhage. Atherosclerosis was extensive in rabbits with serum cholesterol concentrations greater than 300 mg/100 ml and minimal in rabbits with concentrations consistently less than 150 mg/100 ml. Thicker lesions tended to have more atheromas while thinner ones were more fibrous and less fatty. Significant regression of fatty streaks occurred in a group of rabbits fed the atherogenic diet for 2 years and a stock diet during the third year but more advanced lesions did not regress. This study illustrates the importance of time as a variable in atherogenesis, and demonstrates that modest hypercholesterolemia can lead to atherosclerosis in rabbits that is qualitatively unlike atherosclerosis induced in rabbits by cholesterol rich diets and that resembles several stages of human atherosclerosis.
Biochemical and anthropometric determinants of serum beta- and pre-beta-lipoproteins in children. Bogalusa Heart Study.
Authors: Berenson G S , Webber L S , Srinivasan S R , Voors A W , Harsha D W , Dalferes E R
Published: June 2011

A special in-depth substudy was conducted on 388 children from a total biracial (black-white) population, who were stratified on levels of serum beta- and pre-beta-lipoprotein cholesterol to explore factors associated with lipoprotein levels in childhood. Biochemical parameters on venous blood samples were obtained both on fasting subjects and after an abbreviated glucose tolerance test, along with selected anthropometric measures like height, weight, and skinfolds. Biochemical and anthropometric relationships were minimal for children with elevated beta-lipoprotein cholesterol and low pre-beta-lipoprotein cholesterol. On the other hand, children with higher levels of pre-beta-lipoprotein cholesterol, with or without elevated beta-lipoprotein cholesterol, showed associations with fatness and slightly higher levels of glucose and insulin, with other biochemical parameters considered within normal levels. These differences noted among free-living children with different levels of serum lipoproteins provide clues to mechanisms involved in the early natural history of coronary artery disease.
Linoleic acid metabolism and prostaglandin production by cultured bovine pulmonary artery endothelial cells.
Authors: Kaduce T L , Spector A A , Bar R S
Published: June 2011

When bovine pulmonary artery endothelial cells are cultured in a medium supplemented with linoleic acid, their capacity to produce prostacyclin (PGI2) is reduced by about 60%. This reduction occurs when PGI2 formation is stimulated by the addition of either the calcium ionophore A23187 or arachidonic acid. In addition, supplementation with linoleic acid reduced the production of prostaglandin E2 and F2 alpha from 1-14C-arachidonic acid by more than 50%. The capacity of cultured bovine pulmonary vein and aortic endothelial cells to convert extracellular arachidonic acid into PGI2 also was reduced by about 50% when the growth medium was supplemented with linoleic acid. Although bovine pulmonary artery endothelial cells incorporated large amounts of 1-14C-linoleic acid into cellular phospholipids and triglycerides, a maximum of only 2.3% of the radioactivity was converted to arachidonic acid in 24 hours. The most prevalent radioactive metabolite was eicosadienoic acid, the elongation product of linoleic acid. As compared with linoleic acid, the bovine endothelial cells incorporated 30% more 1-14C-arachidonic acid into phospholipids and 60% more into triglycerides. When the growth medium was supplemented with linoleic acid, the percentage of this fatty acid in cellular lipids increased 3- to 4.5-fold and eicosadienoic acid accumulated, accounting for up to 9% of the cellular fatty acids. This increase was accompanied by a 30% to 45% reduction in arachidonic acid. These findings, together with our previous results with human umbilical vein endothelium, suggest that an inability to convert large amounts of linoleic to arachidonic acid and a suppressive effect of linoleic acid enrichment on prostaglandin production may be general properties of endothelial cells.
Stimulation of Phospholipase D Activity by Oxidized LDL in Mouse Peritoneal Macrophages
Authors: Gómez-Muñoz Antonio , Martens Jason S. , Steinbrecher Urs P.
Published: June 2011

Abstract—Oxidation of LDL is an important factor in the development of atherosclerosis. However, the mechanisms by which oxidized LDL exerts its atherogenic actions are poorly understood. In the present work, we show that oxidized LDL stimulates phospholipase D (PLD) activity in mouse peritoneal macrophages and that this effect increases with the degree of LDL oxidation. Oxidative modification of LDL results in the production of lipid peroxides and the conversion of phosphatidylcholine to lysophosphatidylcholine. Although we found that lysophosphatidylcholine alone activates PLD, the stimulation of this enzyme activity by oxidized LDL is independent of lysophosphatidylcholine formation. Also, 7-ketocholesterol, the major oxysterol in oxidized LDL, failed to stimulate PLD activity. To determine the mechanism(s) whereby oxidized LDL activates PLD, the possible involvements of protein kinase C and tyrosine phosphorylation were investigated. Pretreatment of macrophages with the protein kinase C inhibitor Ro-32-0432 or downregulation of protein kinase C activity by prolonged incubation with 100 nmol/L 4β-phorbol 12-myristate 13-acetate did not alter the stimulatory effect of oxidized LDL on PLD activation. However, oxidized LDL stimulated tyrosine phosphorylation of several macrophage proteins, and preincubation of the macrophages with genistein, a tyrosine kinase inhibitor, blocked the activation of PLD by oxidized LDL. In addition, pretreatment with orthovanadate, which inhibits tyrosine phosphatases, enhanced basal and oxidized LDL–stimulated PLD activity. Pretreatment of macrophages with pertussis toxin decreased the stimulatory effect of oxidized LDL, indicating that GTP-binding proteins may also be involved in the activation of PLD by oxidized LDL. We also found that the platelet-activating factor receptor antagonists WEB 2086 and L-659,989 inhibit the oxidized LDL stimulation of PLD, suggesting a role for platelet-activating factor receptor in this process. The stimulation of the PLD pathway by oxidized LDL may be of importance in atherogenesis, because PLD activation leads to generation of important second messengers such as phosphatidate, lysophosphatidate, and diacylglycerol, which are known to regulate many cellular functions.
Oxidized VLDL Induces Less Triglyceride Accumulation in J774 Macrophages Than Native VLDL Due to an Impaired Extracellular Lipolysis
Authors: Jong Miek C. , Hendriks Wendy L. , van Vark Leonie C. , Dahlmans Vivian E. H. , Groener Johanna E. M. , Havekes Louis M.
Published: June 2011

Abstract
—The present study examined the relative contributions of the different pathways by which oxidatively modified VLDL (oxVLDL) promotes the uptake and intracellular accumulation of lipids in J774 macrophages. VLDL was oxidized for a maximum of 4 hours, resulting in an increase in thiobarbituric acid–reactive substances and an increased electrophoretic mobility on agarose gel. The lipid composition of the relatively moderately oxidized VLDL samples did not differ significantly from that of nonoxidized VLDL samples. The uptake of
125
I-labeled VLDL by the J774 cells increased with oxidation time and was completely blocked on coincubation with polyinosinic acid (PolyI), indicating that oxVLDL is taken up by the cells via the scavenger receptor only. Despite the 2-fold increased uptake of oxVLDL protein, the cell association of triglyceride (TG)-derived fatty acids by the J774 macrophages after incubation with oxVLDL was only 50% of that with native VLDL. In line with these observations, the induction of de novo synthesis of TG by J774 cells was ≈3-fold less efficient after incubation with oxVLDL than after incubation with native VLDL. The induction of de novo synthesis of TG with oxVLDL was even further decreased on simultaneous incubation with PolyI, whereas PolyI did not affect the native VLDL-induced TG synthesis. These results indicate that oxVLDL induces endogenous TG synthesis predominantly through particle uptake via the scavenger receptor and much less via the extracellular lipoprotein lipase (LPL)–mediated hydrolysis of TG, as is the case for native VLDL. In line with these observations, we showed that the suitability of VLDL as a substrate for LPL decreases with oxidation time. Addition of oxVLDL to the LPL assay did not interfere with the lipolysis of native VLDL. However, enrichment of the oxidized lipoprotein particle with native apoC2 was able to fully restore the impaired lipolysis. Thus, from these studies it can be concluded that on oxidation, VLDL becomes less efficient in inducing TG accumulation in J774 cells as a consequence of a defect in apoC2 as an activator for the LPL-mediated extracellular lipolysis.
Controlled Trial of α-Tocopherol and β-Carotene Supplements on Stroke Incidence and Mortality in Male Smokers
Authors: Leppälä Jaana M. , Virtamo Jarmo , Fogelholm Rainer , Huttunen Jussi K. , Albanes Demetrius , Taylor Philip R. , Heinonen Olli P.
Published: June 2011

Abstract
—Observational data suggest that diets rich in fruits and vegetables and with high serum levels of antioxidants are associated with decreased incidence and mortality of stroke. We studied the effects of α-tocopherol and β-carotene supplementation. The incidence and mortality of stroke were examined in 28 519 male cigarette smokers aged 50 to 69 years without history of stroke who participated in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC Study). The daily supplementation was 50 mg α-tocopherol, 20 mg β-carotene, both, or placebo. The median follow-up was 6.0 years. A total of 1057 men suffered from incident stroke: 85 men had subarachnoid hemorrhage; 112, intracerebral hemorrhage; 807, cerebral infarction; and 53, unspecified stroke. Deaths due to stroke within 3 months numbered 38, 50, 65, and 7, respectively (total 160). α-Tocopherol supplementation increased the risk of subarachnoid hemorrhage 50% (95% CI −3% to 132%,
P
=0.07) but decreased that of cerebral infarction 14% (95% CI −25% to −1%,
P
=0.03), whereas β-carotene supplementation increased the risk of intracerebral hemorrhage 62% (95% CI 10% to 136%,
P
=0.01). α-Tocopherol supplementation also increased the risk of fatal subarachnoid hemorrhage 181% (95% CI 37% to 479%,
P
=0.01). The overall net effects of either supplementation on the incidence and mortality from total stroke were nonsignificant. α-Tocopherol supplementation increases the risk of fatal hemorrhagic strokes but prevents cerebral infarction. The effects may be due to the antiplatelet actions of α-tocopherol. β-Carotene supplementation increases the risk of intracerebral hemorrhage, but no obvious mechanism is available.
Influence of β0-Thalassemia on the Phenotypic Expression of Heterozygous Familial Hypercholesterolemia
Authors: Deiana Luca , Garuti Rita , Pes Giovanni Mario , Carru Ciriaco , Errigo Alessandra , Rolleri Marina , Pisciotta Livia , Masturzo Paola , Cantafora Alfredo , Calandra Sebastiano , Bertolini Stefano
Published: June 2011

Abstract—One of the genetic features of the Sardinian population is the high prevalence of hemoglobin disorders. It has been estimated that 13% to 33% of Sardinians carry a mutant allele of the α-globin gene (α-thalassemia trait) and that 6% to 17% are β-thalassemia carriers. In this population, a single mutation of β-globin gene (Q39X, β039) accounts for >95% of β-thalassemia cases. Because previous studies have shown that Sardinian β-thalassemia carriers have lower total and low density lipoprotein (LDL) cholesterol than noncarriers, we wondered whether this LDL-lowering effect of the β-thalassemia trait was also present in subjects with familial hypercholesterolemia (FH). In a group of 63 Sardinian patients with the clinical diagnosis of FH, we identified 21 unrelated probands carrying 7 different mutations of the LDL receptor gene, 2 already known (313+1 g>a and C95R) and 5 not previously reported (D118N, C255W, A378T, T413R, and Fs572). The 313+1 g>a and Fs572 mutations were found in several families. In cluster Fs572, the plasma LDL cholesterol level was 5.76±1.08 mmol/L in subjects with β0-thalassemia trait and 8.25±1.66 mmol/L in subjects without this trait (P<0.001). This LDL-lowering effect was confirmed in an FH heterozygote of the same cluster who had β0-thalassemia major and whose LDL cholesterol level was below the 50th percentile of the distribution in the normal Sardinian population. The hypocholesterolemic effect of β0-thalassemia trait emerged also when we pooled the data from all FH subjects with and without β0-thalassemia trait, regardless of the type of mutation in the LDL receptor gene. The LDL-lowering effect of β0-thalassemia may be related to (1) the mild erythroid hyperplasia, which would increase the LDL removal by the bone marrow, and (2) the chronic activation of the monocyte-macrophage system, causing an increased secretion of some cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-α) known to affect the hepatic secretion and the receptor-mediated removal of apolipoprotein B–containing lipoproteins. The observation that our FH subjects with β0-thalassemia trait (compared with noncarriers) have an increase of blood reticulocytes (40%) and plasma levels of interleukin-6 (+60%) supports these hypotheses. The lifelong LDL-lowering effect of β0-thalassemia trait might slow the development and progression of coronary atherosclerosis in FH.
Effect of ω-3 Fatty Acids and Simvastatin on Hemostatic Risk Factors and Postprandial Hyperlipemia in Patients With Combined Hyperlipemia
Authors: Nordøy Arne , Bønaa Kaare H. , Sandset Per Morten , Hansen John-Bjarne , Nilsen Hugo
Published: June 2011

Abstract
—Patients with combined hyperlipemia have lipid abnormalities associated with an increased tendency to develop atherosclerosis and thrombosis. This tendency may be accelerated during postprandial hyperlipemia. In the present double-blind parallel study, 41 patients with combined hyperlipemia and serum triacylglycerols between 2.0 and 15.0 mmol/L and serum total cholesterol >5.3 mmol/L at the end of a 3-month dietary run-in period were treated with simvastatin at 20 mg/d for at least 10 weeks; patients were then randomized into 2 groups receiving simvastatin+ω-3 fatty acids at 3.36 g/d or placebo (corn oil) for an additional 5 weeks. Hemostatic variables that have been associated with increased thrombotic tendency were evaluated with subjects in the fasting state and during postprandial hyperlipemia before and after combined treatment. Supplementation of ω-3 fatty acid reduced tissue factor pathway inhibitor antigen (
P
<0.05) in the fasting state, reduced the degree of postprandial hyperlipemia (
P
<0.005), and reduced activated factor VII concentration appearing during postprandial hyperlipemia. In conclusion, ω-3 fatty acids given in addition to simvastatin to patients with combined hyperlipemia reduced the free tissue factor pathway inhibitor fraction in the fasting state and inhibited the activation of factor VII occurring during postprandial lipemia, thus representing a potential beneficial effect on the hemostatic risk profile in this patient group.
Improvement of Nitric Oxide–Dependent Vasodilatation by HMG-CoA Reductase Inhibitors Through Attenuation of Endothelial Superoxide Anion Formation
Authors: Wagner Andreas H. , Köhler Thomas , Rückschloss Uwe , Just Ingo , Hecker Markus
Published: June 2011

Abstract—Three 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), atorvastatin, pravastatin, and cerivastatin, inhibited phorbol ester–stimulated superoxide anion (O2−) formation in endothelium-intact segments of the rat aorta in a time- and concentration-dependent manner (maximum inhibition of 70% after 18 hours at 1 to 10 μmol/L). The HMG-CoA reductase product mevalonic acid (400 μmol/L) reversed the inhibitory effect of the HCRIs, which, conversely, was mimicked by inactivation of p21 Rac withClostridium sordelliilethal toxin but not by inactivation of p21 Rho withClostridium botulinumexoenzyme (C3). A mevalonate-sensitive inhibition of phorbol ester–stimulated O2−formation by atorvastatin was also observed in porcine cultured endothelial cells and in a murine macrophage cell line. In the rat aorta, no effect of the HCRIs on protein kinase C, NADPH oxidase, or superoxide dismutase (SOD) activity and expression was detected, whereas that of endothelial nitric oxide (NO) synthase was enhanced ≈2-fold. Moreover, exposure of the segments to atorvastatin resulted in a significant improvement of endothelium-dependent NO-mediated relaxation, and this effect was abolished in the presence of SOD. Taken together, these findings suggest that in addition to augmenting endothelial NO synthesis, HCRIs inhibit endothelial O2−formation by preventing the isoprenylation of p21 Rac, which is critical for the assembly of NADPH oxidase after activation of protein kinase C. The resulting shift in the balance between NO and O2−in the endothelium improves endothelial function even in healthy blood vessels and therefore may provide a reasonable explanation for the beneficial effects of HCRIs in patients with coronary heart disease in addition to or as an alternative to the reduction in serum LDL cholesterol.
Cyclic GMP–Dependent Protein Kinase Expression in Coronary Arterial Smooth Muscle in Response to Balloon Catheter Injury
Authors: Anderson Peter G. , Boerth Nancy J. , Liu Ming , McNamara Dennis B. , Cornwell Trudy L. , Lincoln Thomas M.
Published: June 2011

Abstract
—Arterial smooth muscle cells undergo phenotypic and proliferative changes in response to balloon catheter injury. Nitric oxide (NO) and cGMP have been implicated in the inhibition of vascular smooth muscle cell proliferation and phenotypic modulation in cultured-cell studies. We have examined the expression of the major cGMP receptor protein in smooth muscle, cGMP-dependent protein kinase I (PKG), in response to balloon catheter injury in the swine coronary artery. On injury, there was a transient decrease in the expression of PKG in neointimal smooth muscle cells when compared with medial smooth muscle cells. The decrease in PKG expression was observed in the population of proliferating cells expressing the extracellular matrix protein osteopontin but not in cells present in the uninjured portion of the media. Coincident with the suppression of PKG expression in neointimal cells after injury, there was a marked increase in the expression of type II NO synthase (inducible NOS [iNOS], NOS-II) in the neointimal cells. These results suggest that PKG expression is transiently reduced in response to injury in the population of coronary arterial smooth muscle cells that are actively proliferating and producing extracellular matrix proteins. The reduction in PKG expression is also correlated temporally with increases in inflammatory activity in the injured vessels as assessed by iNOS expression. Coupled with our current knowledge regarding the role of PKG in the regulation of cultured cell phenotypes, these results imply that PKG may also regulate phenotypic modulation of vascular smooth muscle cells in vivo as well.
Effect of Fluid Flow on Smooth Muscle Cells in a 3-Dimensional Collagen Gel Model
Authors: Wang Su , Tarbell John M.
Published: June 2011

Abstract
—A 3D collagen gel model was developed to simulate interstitial fluid flow and to assess the importance of this flow on the biochemical production rates of vascular smooth muscle cells (SMCs). Rat aortic SMCs were suspended in type I collagen, and the gel was supported by nylon fibers that allowed a 9-cm length of the SMC-gel model to withstand 90 cm H
2
O differential pressure over a 6-hour period without significant compaction. Up to 1 dyne/cm
2
shear stress on the suspended SMCs could be induced by the pressure-driven interstitial flow. The suspended SMCs were globular, had a diameter of ≈10 μm, and were distributed uniformly throughout the gel. The collagen fibers formed a network that was connected randomly with the surface of SMCs and nylon fibers. The diameter of the collagen fibers was ≈100 nm, and the concentration of collagen was 2.5 mg/mL. Using these parameters, fiber matrix theory predicted a Darcy permeability coefficient (
K
p
) of 1.22×10
−
8
cm
2
, which was close to the measured value of
K
p
. The production rates of prostaglandin (PG) I
2
and PGE
2
were used as markers of biochemical responsiveness of SMCs to fluid shear stress. Both PGI
2
and PGE
2
production rates under 1 dyne/cm
2
shear stress were significantly elevated relative to static (no-flow) controls. The production rates, however, were ≈10 times lower than observed when the same cells were plated on collagen-treated glass slides (2D model) and exposed to the same level of shear stress by use of a rotating disk apparatus. The results indicate that interstitial flow can affect SMC biology and that SMCs are more quiescent in 3D cultures than in 2D cultures. The 3D collagen gel model should be useful for future studies of interstitial flow effects on SMC function.
Acute Antithrombotic Effect of a Front-Loaded Regimen of Clopidogrel in Patients With Atherosclerosis on Aspirin
Authors: Helft Gérard , Osende Julio I. , Worthley Stephen G. , Zaman Azfar G. , Rodriguez Oswaldo J. , Lev Eli I. , Farkouh Michael E. , Fuster Valentin , Badimon Juan J. , Chesebro James H.
Published: June 2011

Abstract
—There is a need for a rapid antithrombotic effect after the administration of antiplatelet drugs in the setting of acute coronary syndromes and percutaneous interventions. Clopidogrel, a new thienopyridine derivative, is an efficient antiplatelet agent. However, the standard regimen of clopidogrel (75 mg/d) requires 2 to 3 days before significant antithrombotic effects. Patients with stable arterial disease on chronic aspirin therapy (n=20) were treated with clopidogrel either with a front-loaded regimen, 300 mg the first day and 75 mg/d the next 7 days, or with a standard regimen, 75 mg/d for 8 days. Blood thrombogenicity was assessed by quantification of platelet-thrombus formation in an ex vivo perfusion chamber, by ADP-induced platelet aggregation, and by ADP-induced fibrinogen binding. At 2 hours, mean total thrombus area with the standard regimen was not significantly reduced. In contrast, at 2 hours, the mean total thrombus area with the front-loaded regimen was significantly decreased by 23.1±8.5% versus baseline (
P
<0.05). ADP-induced platelet aggregation (with 5 and 10 μmol/L) was also significantly (
P
<0.05) reduced with the front-loaded regimen at 2 hours, with the mean platelet aggregation being 82.2±4.4% and 81.8±4.5%, respectively, versus baseline. Similarly, flow cytometry demonstrated a significant decrease (
P
<0.05) in the ADP-induced fibrinogen binding (with 0.12 and 0.6 μmol/L) at 2 hours in this front-loaded regimen group (36.1±2.0% and 53.2±9.3%). With the standard regimen, platelet activity was not significantly reduced at 2 hours. Our data suggest that a front-loaded regimen of clopidogrel added to aspirin achieves a significant antithrombotic effect at 2 hours in patients with known atherosclerotic disease on chronic aspirin therapy. This provides a rationale for using front-loaded clopidogrel in combination with aspirin in percutaneous coronary interventions.
Role of Platelet P-Selectin and CD40 Ligand in the Induction of Monocytic Tissue Factor Expression
Authors: Lindmark Eva , Tenno Taavo , Siegbahn Agneta
Published: June 2011

Abstract—Activated platelets can express CD40 ligand (CD40L) and trigger inflammatory response and tissue factor (TF) expression in endothelial cells through interaction with CD40. This pathway is also important for T cell–induced monocyte and endothelial cell procoagulant activity. We have studied the potential role of the CD40-CD40L pathway in platelet-induced TF expression in a monocytic cell line and in whole-blood monocytes. In vitamin D3–differentiated U-937 cells, thrombin-stimulated platelets increased TF expression as measured by mRNA quantification, flow cytometry, and procoagulant activity. Maximum antigen expression occurred after 2 hours. Neutralizing anti–P-selectin antibody yielded a 50% suppression of procoagulant activity, whereas antibody to CD40L had no effect. In thrombin receptor activator–stimulated citrated blood, monocytes were up to 77% TF-positive, with peak expression after only 15 minutes. However, no TF mRNA was detectable at that time. Anti–P-selectin antibody reduced TF by 50%, whereas antibody to CD40L gave a 17% reduction. Thus, we conclude that P-selectin exposed on activated platelets induces the expression of TF in both U-937 cells and whole-blood monocytes but by different mechanisms. Platelet CD40L does not display any significant effect on U-937 cells but may be of some importance on whole-blood monocytes. This suggests a possible functional difference between U-937 and monocyte CD40. Another important finding in this study is the rapid appearance of surface TF on monocytes without detectable mRNA formation. This indicates that TF may be stored intracellularly in these cells and can be exposed on the surface independent of de novo protein synthesis.
Pulsatile Stretch–Induced Extracellular Signal–Regulated Kinase 1/2 Activation in Organ Culture of Rabbit Aorta Involves Reactive Oxygen Species
Authors: Lehoux Stéphanie , Esposito Bruno , Merval Régine , Loufrani Laurent , Tedgui Alain
Published: June 2011

Abstract
—Increased steady intraluminal pressure in blood vessels activates the extracellular signal–regulated kinase (ERK)1/2 pathway. However, signal transduction of pulsatile stretch has not been elucidated. Using an organ culture model of rabbit aorta, we studied ERK1/2 activation by pulsatility in vessels maintained at 80 mm Hg for 24 hours. ERK1/2 activity was evaluated by in-gel kinase assays and by Western blot. Compared with control aortas without pulsatility, aortas submitted to a pulsatile 10% variation in vessel diameter displayed a significant increase in ERK1/2 activity (207±12%,
P
<0.001), which remained high after removal of the endothelium. Unlike steady overstretch, pulsatile stretch–induced activation of ERK1/2 was not modified by herbimycin A, a Src family tyrosine kinase inhibitor, but was reduced by other tyrosine kinase inhibitors, tyrphostin A48 and genistein (162±27% and 144±14%, respectively). Conversely, ERK1/2 activity was markedly decreased in pulsatile vessels treated with staurosporine (114±18%) although neither of the more specific protein kinase C inhibitors, Ro-31-8220 or Gö-6976, blocked ERK1/2 activation (209±24% and 238±34%, respectively), whereas staurosporine had no effect on steady overstretch–induced ERK1/2 activation. Pulsatility induced superoxide anion generation, which was prevented by the NADPH oxidase inhibitor diphenyleneiodonium. Furthermore, polyethylene glycol–superoxide dismutase completely abolished ERK1/2 activation by pulsatility (114±12%). Finally, ERK1/2 and O
2
−
levels in freshly isolated vessels were equivalent to the levels found in pulsatile vessels. In conclusion, pulsatile stretch activates ERK1/2 in the arterial wall via pathways different from those induced by steady overstretch. Pulsatility might be considered a physiological stimulus that maintains a certain degree of ERK1/2 activation via oxygen-derived free radical production.
Interleukin-1 Receptor Antagonist Expression in Human Endothelial Cells and Atherosclerosis
Authors: Dewberry Rachael , Holden Hazel , Crossman David , Francis Sheila
Published: June 2011

Abstract—The proinflammatory cytokine interleukin (IL)-1 is expressed mainly within the endothelium of atherosclerotic plaques and may be linked with inflammatory mechanisms of atherogenesis. IL-1 action is complex and regulated in part by its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1ra). Therefore, we studied differential and specific isoform expression of IL-1ra in the endothelium of diseased coronary arteries and in endothelial cells (ECs) stimulated under defined conditions. In view of an association with IL-1ra gene (IL-1RN) polymorphism, the influence of endothelial cell genotype at IL-1RN on IL-1ra protein production was also examined. Secreted IL-1ra and intracellular IL-1ra mRNAs were detected by semiquantitative reverse transcription–polymerase chain reaction in human atherosclerotic and dilated cardiomyopathic coronary arteries; protein expression appeared increased in atherosclerotic compared with dilated cardiomyopathic arteries, where IL-1ra appeared to be confined to the endothelium. Only intracellular IL-1ra type I mRNA was detected in human umbilical vein ECs (HUVECs) and human coronary artery ECs (HCAECs) when they were stimulated with bacterial lipopolysaccharide/phorbol myristate acetate and transforming growth factor-β. IL-1β and IL-1α were without effect. IL-1ra protein was detected in HUVECs (intracellular IL-1ra), HCAECs (intracellular IL-1ra), and human coronary artery smooth muscle cells (intracellular IL-1ra) by immunoprecipitation and Western blot. IL-1ra was detected in HUVEC cell lysates by ELISA and appeared to be influenced by the genotype of the IL-1RN variable number tandem repeat, an 86-bp repeat polymorphism in intron 2 of the IL-1ra gene, with lower levels of IL-1ra produced by IL-1RN allele 2–containing cells (ratio of IL-1ra to total protein: for 1,1 homozygotes, 1.38±0.28×10−9[n=15]; for 1,2 heterozygotes, 0.81±0.17×10−9[n=8]; and for 2,2 homozygotes, 0.63±0.19×10−9[n=5];P<0.05 compared with 1,1 homozygotes). This is the first demonstration of IL-1ra in human diseased arteries, stimulated HUVECs, and HCAECs and indicates the endothelial cell as an important source. Endothelial IL-1ra production may be controlled by the endothelial IL-1RN genotype. These data further support the role of the IL-1 system of cytokines in the pathogenesis of atherosclerosis.
Paraoxonase (PON1) Phenotype Is a Better Predictor of Vascular Disease Than Is
PON1
192
or
PON1
55
Genotype
Authors: Jarvik Gail Pairitz , Rozek Laura S. , Brophy Victoria H. , Hatsukami Thomas S. , Richter Rebecca J. , Schellenberg Gerard D. , Furlong Clement E.
Published: June 2011

Abstract
—The paraoxonase (PON1)
PON1-Q192R
and
PON1-L55M
polymorphisms have been inconsistently associated with vascular disease. Plasma PON1 activity phenotypes vary markedly within genotypes and were, therefore, expected to add to the informativeness of genotype for predicting vascular disease. The case-control sample included 212 age- and race-matched men (mean age 66.4 years). The 106 carotid artery disease (CAAD) cases had >80% carotid stenosis, and the 106 controls had <15%. Two PON1 substrate hydrolysis rates (paraoxon [POase] and diazoxon [DZOase]) were significantly lower in cases than in controls and were significant predictors of CAAD by use of logistic regression (POase,
P
=0.005; DZOase,
P
=0.019). DZOase predicted vascular disease independently of lipoprotein profile, high density lipoprotein subfractions, apolipoprotein A-I, and smoking.
PON1-192
and
PON1-55
genotypes or haplotypes did not predict case-control status unless the activity phenotype was also included as a predictor by use of logistic regression. When phenotype was included as a predictor,
PON1-192
and
PON1-55
genotypes or combined haplotypes were significant predictors (
P
<0.05). In conclusion, examining
PON1-192
and/or
PON1-55
genotypes alone may mistakenly lead to the conclusion that there is no role of PON1 in CAAD. These results support the benefit of a “level crossing” approach that includes intervening phenotypes in the study of complexly inherited disease.
Platelets From a Patient Heterozygous for the Defect of P2
CYC
Receptors for ADP Have a Secretion Defect Despite Normal Thromboxane A
2
Production and Normal Granule Stores
Authors: Cattaneo Marco , Lecchi Anna , Lombardi Rossana , Gachet Christian , Zighetti Maddalena L.
Published: June 2011

Abstract
—Two unrelated patients with a congenital bleeding diathesis associated with a severe defect of the platelet ADP receptor coupled to adenylate cyclase (P2
CYC
) have been described so far. In one of them, platelet secretion was shown to be abnormal. We recently showed that platelets with the primary secretion defect (PSD; characterized by abnormal secretion but normal granule stores, thromboxane A
2
production, and ADP-induced primary wave of aggregation) have a moderate defect of P2
CYC
. Therefore, the interaction of ADP with the full complement of its receptors seems to be essential for normal platelet secretion, and PSD patients may be heterozygotes for the congenital severe defect of P2
CYC
. In this study, we describe 2 new related patients with a severe defect of P2
CYC
and the son of one of them, who is to be considered an obligate heterozygote for the defect. The 2 patients with the severe defect had lifelong histories of abnormal bleeding, prolonged bleeding times, abnormalities of platelet aggregation and secretion, lack of inhibition of adenylate cyclase by ADP, and a deficiency of platelet-binding sites for [
33
P]2 MeS-ADP (240 and 225 sites per platelet; normal range, 530 to 1102). The son of one of them had a mildly prolonged bleeding time and abnormalities of platelet aggregation and secretion similar to those found in patients with PSD. In addition, his platelets showed a moderate defect of binding sites for [
33
P]2 MeS-ADP (430 sites per platelet) and of adenylate cyclase inhibition by ADP. This study of a family with the platelet disorder characterized by a defect of the platelet P2
CYC
receptor supports our hypothesis that the full complement of the platelet ADP receptors is essential for normal platelet secretion and that some patients with the common, ill-defined diagnosis of PSD are actually heterozygous for the defect.
Neutrophil, Not Macrophage, Infiltration Precedes Neointimal Thickening in Balloon-Injured Arteries
Authors: Welt Frederick G. P. , Edelman Elazer R. , Simon Daniel I. , Rogers Campbell
Published: June 2011

Abstract
—Macrophages are abundant after stent-induced arterial injury. Inhibition of macrophage recruitment blocks neointimal growth in this model. In contrast, after superficial injury from balloon endothelial denudation, macrophages are sparse. However, many anti-inflammatory therapies remain effective against neointimal growth after balloon injury. To investigate further the role of leukocytes after injury, 41 New Zealand White rabbits underwent iliac artery balloon denudation. In 18, subcutaneous pumps were placed to deliver intravenous heparin (0.3 mg/kg per hour). Arteries were harvested at 6 hours and at 3, 7, and 14 days. In 8 animals, either M1/70 (a monoclonal antibody [mAb] against adhesion molecule Mac-1) or a nonspecific IgG was given (5 mg/kg IV bolus and then 1 mg/kg SC QOD), and arteries were harvested at 6 hours and 3 days. Computer-aided morphometry was performed as was immunohistochemistry to assess smooth muscle cell (SMC) proliferation (bromodeoxyuridine-positive cells), neutrophil content (RPN357, mAb against rabbit neutrophil/thymocyte), and macrophage content (RAM-11, mAb against rabbit macrophage). Heparin inhibited neointimal growth at 7 and 14 days (64% and 32.5% reduction, respectively;
P
<0.05). Neutrophils were observed in the media early after balloon injury, and heparin and M1/70 inhibited this infiltration (82% and 83% reduction, respectively;
P
<0.05 each) with a coincident inhibition of medial SMC proliferation at 3 days (49% and 84% reduction, respectively;
P
<0.05 each). Macrophages were absent at all time points. Neutrophil, but not macrophage, infiltration occurs early after endothelial denudation. Inhibition of this process is associated with a reduction in medial SMC proliferation. These data suggest a central role for neutrophils in restenosis and help to explain prior reports of an inhibitory effect of anti-inflammatory therapies on neointimal growth after balloon injury.
Involvement of Oxysterols and Lysophosphatidylcholine in the Oxidized LDL–Induced Impairment of Serum Albumin Synthesis by HEPG2 Cells
Authors: Bourdon Emmanuel , Loreau Nadine , Davignon Jean , Bernier Lise , Blache Denis
Published: June 2011

Abstract
—Oxidized low density lipoproteins (Ox-LDLs) are increasingly thought to be a key element in atherogenesis. We have previously reported that serum albumin has important antioxidant properties and that a reduced synthesis of albumin may represent a crucial point in the overall antioxidant defense. In the present work, we aimed at determining whether Ox-LDL could modulate albumin synthesis in cultured human hepatocytes (HepG2 cells). With the use of enzyme immunoassay and radiolabeled leucine incorporation followed by specific immunoprecipitation, Ox-LDL was found to lead to a dose-dependent decrease in albumin secretion. Moreover, the protein synthesis and mRNA levels were decreased in the presence of Ox-LDL, as assessed by Northern blot analysis. Because oxysterols and lysophospholipids are key components of Ox-LDL, we tested the effects of oxysterols (7-ketocholesterol and 25-hydroxycholesterol) and lysophosphatidylcholine on albumin secretion and expression. In our experimental conditions, we found that incubations with oxysterols or lysophosphatidylcholine at pathophysiological concentrations similar to those measured in Ox-LDLs reproduced the above-mentioned inhibitory effects on albumin synthesis. On the basis of our in vitro data, we propose that this newly described biological effect of Ox-LDL might partly explain the findings of epidemiological studies indicating that reduced levels of serum albumin are associated with increased mortality.