bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Fish Oil on Oxidation Resistance of VLDL in Hypertriglyceridemic Patients
Authors: Hau Man-Fai , Smelt Augustinus H.M. , Bindels Alexander J.G.H. , Sijbrands Eric J.G. , Van der Laarse Arnoud , Onkenhout Willem , van Duyvenvoorde Wim , Princen Hans M.G.
Published: June 2011

In hypertriglyceridemic (HTG) patients the addition of fish oil to the diet causes a marked reduction in the concentration of triglyceride-rich lipoproteins in the serum. To investigate the effects of fish oil on the oxidation resistance of VLDL and LDL in HTG patients, nine male patients received 1 g/d fish oil (containing 55.7% n-3 polyunsaturated fatty acids [PUFAs] and 1 U α-tocopherol/g fish oil) for 6 weeks followed by 5 g/d fish oil for an additional 6 weeks. Cu
2+
-induced oxidation of VLDL and LDL was measured by continuous monitoring of conjugated dienes. Supplementation with 1 g/d fish oil caused hardly any changes in the n-3 PUFA content of lipoproteins or lipoprotein concentrations in serum. However, supplementation with 5 g/d fish oil resulted in a significant increase of n-3 PUFA content in VLDL (from 2.5% to 6.4% of total fatty acids) and LDL (from 3.2% to 6.4% of total fatty acids), decreases in serum triglyceride, VLDL triglyceride, and VLDL cholesterol concentrations of 54%, 56%, and 40%, respectively, and an increase in LDL cholesterol of 23%. The lag times of VLDL and LDL oxidation decreased from 197 to 140 minutes (−29%) and 101 to 86 minutes (−15%), respectively. At the end of the 5 g/d fish oil supplementation the lag times of VLDL and LDL oxidation were correlated with their respective n-3 PUFA content (
r
=−.67;
P
<.05 and
r
=−.79;
P
<.02, respectively). Before and at the end of supplementation with 5 g/d fish oil the lag times and propagation rates of VLDL oxidation also correlated with the total number of double bonds in all PUFAs of VLDL. We conclude that fish oil supplementation strongly reduces serum concentrations of total triglycerides, VLDL triglycerides, and VLDL cholesterol. However, in HTG patients fish oil supplementation increased the serum LDL cholesterol concentration and the susceptibility of VLDL and LDL to oxidation.
“Tall Oil”–Derived Phytosterols Reduce Atherosclerosis in ApoE-Deficient Mice
Authors: Moghadasian Mohammed H. , McManus Bruce M. , Pritchard P. Haydn , Frohlich Jiri J.
Published: June 2011

We investigated the effects of a “tall oil”–derived phytosterol mixture (TODPM) on the formation of atherosclerotic lesions in apoE-deficient mice. TODPM was added at 2% (wt/wt) to the chow of nine mice; the control group had six animals. The diet of all animals contained 9% (wt/wt) fat and 0.15% (wt/wt) cholesterol. After 4 weeks, plasma total cholesterol levels were significantly reduced in the TODPM-treated mice (26.6 versus 42.0 mmol/L,
P
<.0001). The mean body weight of the TODPM-supplemented group was significantly higher at week 5 and throughout the study (29.4 versus 27.7 g,
P
<.05). The experiment was terminated at 18 weeks. Histological examination showed mature atherosclerotic lesions composed of foam cells underlying the endothelium, a mosaic of extracellular glycosaminoglycans, numerous apparently proliferative smooth muscle cells, and foci of cholesterol clefts in the control animals. By contrast, the TODPM-treated mice showed only early lesions containing mainly superficial foam cells. As assessed by morphometry, the lesion area in the aortic sinuses of TODPM-treated animals was less than half that of control animals (
P
<.0001). This reduced lesion area was accompanied by a substantial reduction in all lesional components, reflecting a delay in progression of atheromatous changes. A strong positive correlation (
r
=.69,
P
<.01) was found between plasma total cholesterol levels and lesion area in the aortic sinuses. TODPM also prevented the occurrence of xanthomatosis. We conclude that supplementation of a cholesterol-enriched diet with TODPM significantly lowers plasma cholesterol and retards development of atherosclerosis in apoE-deficient mice, suggesting a therapeutic potential for the mixture of phytosterols studied.
Oxidized LDL Stimulates Mitogen-Activated Protein Kinases in Smooth Muscle Cells and Macrophages
Authors: Kusuhara Masatoshi , Chait Alan , Cader Angelina , Berk Bradford C.
Published: June 2011

It has been proposed that oxidized LDL is more atherogenic than native LDL. However, the mechanisms by which native LDL and oxidized LDL alter function of cells in the vessel wall remain undefined. A signal transduction pathway that mediates many changes in cell function is the mitogen-activated protein (MAP) kinase cascade. We therefore examined the effect of native LDL and oxidized LDL on MAP kinase activity in cultured vascular smooth muscle cells (VSMC), endothelial cells, and macrophages by using an in-gel-kinase assay and anti-phosphotyrosine MAP kinase antibodies. Native LDL and LDL oxidized by the addition of Cu2+(Cu2+-oxidized LDL) stimulated MAP kinase in a time- and dose-dependent manner in baboon and rat VSMC but not in bovine endothelial cells. Cu2+-oxidized LDL stimulated MAP kinase in human monocyte-derived macrophages, but the effect was much greater in cells cultured for 7 days compared with 1 day, suggesting dynamic regulation of the cellular response to oxidized LDL. In rat VSMC, the maximal MAP kinase response to Cu2+-oxidized LDL was significantly greater than the response to native LDL. Cu2+-oxidized LDL was more potent, with half-maximal activation at 15 μg/mL versus 30 μg/mL for native LDL. Stimulation of MAP kinase appeared to involve protein kinase C, since phorbol ester pretreatment for 24 hours blocked MAP kinase activation. Oxidation of LDL by other methods showed that activation of MAP kinase was not well correlated with lipid peroxides or aldehydes, suggesting that other components present in oxidized LDL were responsible. The active moiety appeared to be lipid based on extraction of oxidized LDL with organic solvents. These data indicate that LDL stimulates MAP kinase in VSMC, oxidation of LDL potentiates the effect, a lipid moiety is involved, and Cu2+-oxidized LDL activation of MAP kinase is cell-type specific. These findings suggest a role for MAP kinase in the pathways by which oxidized LDL contributes to altered cellular function associated with atherogenesis.
Possible Functional Interactions of Apolipoprotein B-100 Segments That Associate With Cell Proteoglycans and the ApoB/E Receptor
Authors: Olsson Urban , Camejo Germán , Hurt-Camejo Eva , Elfsber Karin , Wiklund Olof , Bondjers Göran
Published: June 2011

The interaction of apoE lipoproteins with cells appears to be mediated by an association with basic sequences of proteoglycans and the apoB/E receptor. ApoB-100 has basic sequences, homologous with those of apoE, that form part of the apoB/E receptor–binding domain. These sequences of apoB-100 also interact with proteoglycans. We investigated whether such segments, in analogy with apoE, could act cooperatively on LDL interactions with proteoglycans and the receptor. As a model we used the two most basic regions of apoB-100, 3147 through 3157 and 3359 through 3367, connected by three glycines (3145-3157–GGG–3359-3367). Such segments may be proximal in LDL by the presence of a disulfide bridge between Cys(3167) and Cys(3297). The apoB heterodimer but not the separated monomers inhibited
125
I-LDL degradation in fibroblasts and THP-1 cells by 50% at ≈11 μmol/L. The heterodimer affinity with arterial proteoglycans was closer to that of LDL and higher than that of the individual peptides. The heterodimer appears to bind specifically to THP-1 cells, with a
K
d
of 6.2×10
−8
mol/L and a B
max
of 1.3×10
6
molecules/cell. Monoclonal antibody C-7, which recognizes the apoB receptor, inhibited the binding to cells. Treatment of fibroblasts with chondroitinase ABC or chlorate decreased
125
I-LDL degradation markedly. Hydrolysis of pericellular proteoglycans of fibroblasts by chondroitinases reduced mostly the low-affinity, high-capacity component of LDL binding. This compartment appears to hold 70% of the cell-associated LDL when internalization is inhibited at 4°C. Therefore, cell-surface chondroitin sulfate/dermatan sulfate proteoglycans appear to modulate binding and receptor-mediated internalization of LDL. This may be caused, at least in part, by the association of proteoglycans with the apoB-100 segments 3145 through 3157 and 3359 through 3367.
Plasma Kinetics of Cholesteryl Ester Transfer Protein in the Rabbit
Authors: McPherson Ruth , Lau Paulina , Kussie Paul , Barrett Hugh , Tall Alan R.
Published: June 2011

The plasma kinetics of recombinant human cholesteryl ester transfer protein (rCETP) were studied in six rabbits before and after cholesterol feeding (0.5% wt/wt). The rCETP, labeled with the use of the Bolton Hunter reagent, was shown to retain neutral lipid transfer activity. After intravenous infusion, labeled rCETP associated with rabbit lipoproteins to an extent similar to endogenous rabbit CETP (62% to 64% HDL associated). The plasma kinetics of CETP, modeled with the use of SAAM-II, conformed to a two-pool model, likely representing free and loosely HDL-associated CETP (fast pool) and a tightly apo (apolipoprotein) AI–associated (slow pool) CETP. The plasma residency time (chow diet) of the fast pool averaged 7.1 hours and of the slow pool, 76.3 hours. The production rate (PR) into and the fractional catabolic rate (FCR) of the fast pool were 20 and 10 times the PR and FCR, respectively, of the slow pool. In response to cholesterol feeding, CETP PR, FCR, and plasma mass increased by 416%, 60%, and 230%, respectively. There was a strong correlation (
r
=.95,
P
=.003) between the increase in rabbit plasma CETP and the modeled increase in CETP PR in response to cholesterol feeding, suggesting that labeled human rCETP is a satisfactory tracer for rabbit plasma CETP. CETP is catabolized by distinct pools, likely corresponding to an apo AI–associated (slow) pool and a free and/or loosely HDL-associated (fast) pool. Factors that alter the affinity of CETP for HDL would be predicted to result in altered CETP catabolism. The effect of dietary cholesterol on plasma CETP mass can be explained largely by the effects on CETP synthesis, consistent with the observed effects of cholesterol on tissue mRNA levels.
Effect of the ACAT Inhibitor CL 277,082 on Apolipoprotein B48 Transport in Mesenteric Lymph and on Plasma Clearance of Chylomicrons and Remnants
Authors: Martins I.J. , Mortimer B.-C. , Redgrave T.G.
Published: June 2011

Inhibitors of acyl CoA:cholesterol acyltransferase (ACAT) activity previously have been found to decrease the absorption of cholesterol and to be effective antiatherosclerotic agents. Effects on chylomicron (CM) transport could contribute to these effects. No previous study has examined the effect of inhibition of ACAT activity on the intestinal lymph output of apolipoprotein (apo) B48 or on the clearance from plasma of lymph CM. In this study, we selected 2,4-difluoro-phenyl-
N
[[4-(2,2-dimethylpropyl)phenyl]methyl]-
N
-(hepthyl)urea (CL 277,082) to inhibit intestinal ACAT activity and measured its effects on the output of lipids and apo B48 in intestinal lymph. Compared with control untreated rats, treatment with CL 277,082 decreased the lymph outputs of apo B48 and triglyceride. Associated with the effects on transport, the lymph CM were smaller in diameter in rats treated with CL 277,082. The unesterified cholesterol content of lymph CM was markedly increased and the cholesteryl ester (CE) content was decreased. The contents of triglyceride were decreased and phospholipid was increased. Labeled CM were prepared by feeding donor rats with a test meal containing
3
H-cholesterol and
14
C–fatty acid. Traced by the CE label in lymph CM in both control rats and rats treated with CL 277,082, the remnants derived after intravenous injection of CM from rats treated with CL 277,082 were cleared significantly more slowly than CM from untreated rats. Moreover, less CE label was recovered in the livers of both groups of rats after injection of CM from rats treated with CL 277,082. Recovery in the spleen was significantly higher in recipient rats injected with CM from rats treated with CL 277,082 when compared with injections of CM obtained from untreated rats. We conclude that the metabolism of CM is affected by treatment with CL 277,082, partly due to the changes in lymph CM composition and partly due to other effects on the recipient rat.
Medroxyprogesterone Acetate Antagonizes Inhibitory Effects of Conjugated Equine Estrogens on Coronary Artery Atherosclerosis
Authors: Adams Michael R. , Register Thomas C. , Golden Deborah L. , Wagner Janice D. , Williams J. Koudy
Published: June 2011

Although estrogen replacement therapy is associated with reduced risk of coronary heart disease and reduced extent of coronary artery atherosclerosis, the effects of combined (estrogen plus progestin) hormone-replacement therapy are uncertain. Some observational data indicate that users of combined hormone replacement consisting of continuously administered oral conjugated equine estrogens (CEE) and oral sequentially administered (7 to 14 days per month) medroxyprogesterone acetate (MPA) experience a reduction in risk similar to that of users of CEE alone. However, the effects of combined, continuously administered CEE plus MPA (a prescribing pattern that has gained favor) on the risk of coronary heart disease or atherosclerosis are not known. We studied the effects of CEE (monkey equivalent of 0.625 mg/d) and MPA (monkey equivalent of 2.5 mg/d), administered separately or in combination, on the extent of coronary artery atherosclerosis (average plaque size) in surgically postmenopausal cynomolgus monkeys fed atherogenic diets and treated with these hormones for 30 months. Treatment with CEE alone resulted in atherosclerosis extent that was reduced 72% relative to untreated (estrogen-deficient) controls (P<.004). Atherosclerosis extent in animals treated with CEE plus MPA or MPA alone did not differ from that of untreated controls. Although treatment had marked effects on plasma lipoprotein patterns, statistical adjustment for variation in plasma lipoproteins did not alter the between-group relationships in atherosclerotic plaque size, suggesting that these factors do not explain substantially the atheroprotective effect of estrogen or the MPA-associated antagonism. Although the mechanism(s) remains unclear, we conclude that oral CEE inhibits the initiation and progression of coronary artery atherosclerosis and that continuously administered oral MPA antagonizes this atheroprotective effect.
Distal Apolipoprotein C-III Regulatory Elements F to J Act as a General Modular Enhancer for Proximal Promoters That Contain Hormone Response Elements
Authors: Kardassis Dimitris , Tzameli Iphigenia , Hadzopoulou-Cladaras Margarita , Talianidis Iannis , Zannis Vassilis
Published: June 2011

Transient transfection assays have shown that the distal apoC-III promoter segments that contain the regulatory elements F to J enhance the strength of the tandemly linked proximal apoA-I promoter 5- to 13-fold in hepatic (HepG2) cells. Activation in intestinal (CaCo-2) cells to levels comparable to those obtained in HepG2 cells requires a larger apoA-I promoter sequence that extends to nucleotide −1500 as well as the presence of hepatic nuclear factor-4 (HNF-4). The distal apoC-III regulatory elements can also enhance 4- to 8-fold the strength of the heterologous apoB promoter in HepG2 and CaCo-2 cells. Finally, these elements in the presence of HNF-4 enhance 14.5- to 18.5-fold the strength of the minimal adenovirus major late promoter linked to two copies of the hormone response element (HRE) AID of apoA-I in both HepG2 and CaCo-2 cells. In vitro mutagenesis of the promoter/enhancer cluster established that the enhancer activity is lost by a mutation in the HRE present in the 3′ end of the regulatory element I (−736 to −714) and is reduced significantly by point mutations or deletions in one or more of the regulatory elements F to J of the apoC-III enhancer. The enhancer activity also requires the HREs of the proximal apoA-I promoter. The apoC-III enhancer can also restore the activity of the proximal apoA-I and apoB promoters that have been inactivated by mutations in CCAAT/enhancer binding protein binding sites, indicating that C/EBP may not participate in the synergistic activation of the promoter/enhancer cluster. The findings suggest that the regulatory elements F to J of the apoC-III promoter act as a general modular enhancer that can potentiate the strength of proximal promoters that contain HREs. Such potentiation in the HepG2 cells can be accounted for by synergistic interactions between HNF-4 or other nuclear hormone receptors bound to the proximal and distal HREs and SP1 or other factors bound to the apoC-III enhancer. Additional factors may be required for optimal activity in CaCo-2 cells as well as for the function of this region as an intestinal enhancer.
Studies on the Mechanism of Fibrate-Inhibited Expression of Plasminogen Activator Inhibitor-1 in Cultured Hepatocytes From Cynomolgus Monkey
Authors: Arts Janine , Kockx Maaike , Princen Hans M.G. , Kooistra Teake
Published: June 2011

Fibrates are widely used drugs in hyperlipidemic disorders. In addition to lowering serum triglyceride levels, fibrates have also been shown to reduce elevated plasma plasminogen activator inhibitor-1 (PAI-1) levels in vivo. We demonstrate that fibrates suppress PAI-1 synthesis in cultured cynomolgus monkey hepatocytes in a concentration-dependent way (0.1 to 1.0 mmol/L) and independent of their lipid-lowering effect. Different fibrates showed different potency in suppressing PAI-1 production: gemfibrozil and clofibric acid, at a concentration of 1 mmol/L, reduced PAI-1 synthesis over 24 hours to 52±20% and 60±5%, while clofibrate and bezafibrate lowered PAI-1 synthesis to only 86±17% and 84±15% of control values, respectively. These changes in PAI-1 production by fibrates correlated with changes in PAI-1 mRNA levels and were also visible at the level of gene transcription. Fibrates did not lower basal PAI-1 synthesis but attenuated an acceleration of PAI-1 production during culture. The suppressing effect of fibrates on PAI-1 synthesis could not be mimicked with activators or inhibitors of protein kinase C (PKC). Furthermore, fibrates did not inhibit the increase in PAI-1 synthesis induced by epidermal growth factor or transforming growth factor-β. These results make mechanisms involving PKC modulation or growth factor receptor inactivation as a mode of action of fibrates unlikely. The suppressing effect of fibrates on PAI-1 synthesis could involve the nuclear receptor peroxisome proliferator–activated receptor (PPAR) and its heterodimeric partner, the retinoid X receptor (RXR). The alpha forms of PPAR and RXR were both found to be expressed in cynomolgus monkey hepatocytes. The ligand for RXRα, 9-
cis
retinoic acid, suppressed PAI-1 synthesis to the same extent as gemfibrozil, while a combination of gemfibrozil and 9-
cis
retinoic acid had no more effect on PAI-1 synthesis than any of these compounds alone at optimal concentrations. In conclusion, fibrates downregulate an induced PAI-1 production in cynomolgus monkey hepatocytes independent of a decrease in triglyceride levels. A possible involvement of PPARα/RXRα in this downregulation is discussed.
Plasminogen Activator Inhibitor-1 Promoter 4G/5G Genotype and Plasma Levels in Relation to a History of Myocardial Infarction in Patients Characterized by Coronary Angiography
Authors: Ossei-Gerning Nicholas , Mansfield Michael W. , Stickland Max H. , Wilson Ian J. , Grant Peter J.
Published: June 2011

To investigate the relationship between an insertion/deletion (4G/5G) polymorphism in the promoter region of the plasminogen activator inhibitor-1 (PAI-1) gene and the phenotypes of PAI-1 levels, coronary atheroma, and a past history of coronary thrombosis, we studied 453 patients (320 men and 133 women) characterized by coronary angiography. Patients were classified as having normal vessels (n=125) or single-vessel (n=92) or multivessel (n=232) coronary disease on the basis of ≥50% stenosis. PAI-1 antigen levels were highest in patients with the 4G/4G genotype (22.5 ng/mL), with a stepwise decrease in levels as the number of 4G alleles decreased (21.5 ng/mL for 4G/5G and 15.8 ng/mL for 5G/5G,
P
=.02) after adjusting for age, sex, triglyceride levels, and body mass index (BMI). The association between triglyceride level and PAI-1 was genotype specific, with a steeper slope in subjects with the 4G/4G genotype (
P
=.004). A gene-environment interaction between BMI, PAI-1, and genotype was observed, with a steeper association in patients with the 5G/5G genotype (
P
=.02). The 4G/4G genotype was significantly associated with a history of myocardial infarction (
P
<.03; odds ratio, 2.0; 95% CI, 1.1 to 3.7). This relationship was stronger in subjects with diseased vessels (
P
=.006). There was no relationship between either PAI-1 genotype or levels and the presence of atheroma. Our data suggest that PAI-1 promoter polymorphism influences the development of myocardial infarction through its effect on thrombus formation in patients with preexisting coronary atheroma.
Polymorphism of the Apolipoprotein E Gene and Early Carotid Atherosclerosis Defined by Ultrasonography in Asymptomatic Adults
Authors: Cattin L. , Fisicaro M. , Tonizzo M. , Valenti M. , Danek G.M. , Fonda M. , Da Col P.G. , Casagrande S. , Pincetti E. , Bovenzi M. , Baralle F.
Published: June 2011

Clinical and autoptical studies have suggested a predisposing role of the allele E4 of apolipoprotein E (apoE) in the development of atherosclerosis and cardiovascular disease. To investigate the possible contribution of apoE allele polymorphism to the carotid intima-media thickness (IMT) as assessed by ultrasound, we studied 260 asymptomatic nondiabetic subjects (121 men, 139 women; mean±SD age, 53±7 years), randomly selected from the population register of the inhabitants of Trieste, Italy. B-mode ultrasound was used to quantify the maximum IMT at 12 sites on the near and far wall of the common, bifurcation, and internal carotid arteries. ApoE genotypes were determined from amplified apoE sequences by restriction isotyping. The frequencies of E2, E3, and E4 alleles were 0.073, 0.827, and 0.100, respectively. As expected, subjects with E4 allele had the highest levels of total serum cholesterol and LDL cholesterol, subjects with E2 allele had the lowest levels, and those with E3 genotype had intermediate levels. The echographic measurements of carotid IMT showed increasing values from E2 to E4 carriers. After adjustment for total and LDL cholesterol serum levels, triglycerides, ratio of LDL to HDL cholesterol, age, sex, and body mass index, ANCOVA showed that the common carotid IMT was significantly greater (
P
=.029) in subjects with E4 allele compared with E3 carriers. Our data confirm the influence of apoE4 on cholesterol levels and clearly show that apoE genotype affects carotid atherosclerosis in its early stages in middle-aged asymptomatic subjects.
Redistribution of von Willebrand Factor in Porcine Carotid Arteries After Balloon Angioplasty
Authors: Giddings J.C. , Banning A.P. , Ralis H. , Lewis M.J.
Published: June 2011

Abstract
von Willebrand factor (VWF) is a well-characterized multimeric glycoprotein present in platelets and plasma and synthesized by vascular endothelial cells and megakaryocytes. Its role in platelet-vessel wall interactions has been studied extensively, but its involvement in intravascular events after balloon angioplasty has not been clarified. VWF antigen is not present in porcine arterial endothelium (except for the pulmonary artery) but is readily detected in porcine venous endothelial cells. We have examined the localization of VWF in porcine vessel walls during neointima formation after bilateral carotid balloon-angioplasty. Endothelium was denuded by balloon injury but regenerated by 7 days and was fully confluent by 42 days. VWF was detected at the site of injury in localized, adherent platelet aggregates at 10 minutes after angioplasty that were not present at later time points. A well-demarcated homogeneous layer of VWF was observed on the luminal surface from 30 minutes to day 7, but there was a progressive shift of positive staining from the lumen to the outer media from days 1 to 7. VWF was also strongly detected at sites proximal and distal to the balloon injury from 30 minutes to day 7, although endothelial disruption was minimal and the monolayer remained substantially intact at these sites. Regrowing endothelial cells appeared to contain granular VWF from days 12 to 21, but this was not readily evident at later time points. The results suggest that balloon injury is associated with deposition and medial absorption of plasma or platelet VWF in this porcine model over a time period that precedes and overlaps vascular smooth muscle proliferation and endothelial recoverage. The findings provide evidence to support the concept of a wider role for VWF in tissue injury responses.
Dysregulation of Monocytic Nuclear Factor-κB by Oxidized Low-Density Lipoprotein
Authors: Brand Korbinian , Eisele Tamara , Kreusel Ursula , Page Michael , Page Sharon , Haas Monika , Gerling Astrid , Kaltschmidt Christian , Neumann Franz-Josef , Mackman Nigel , Baeuerle Patrick A. , Walli Autar K. , Neumeier Dieter
Published: June 2011

AbstractNuclear factor-κB (NF-κB)/Rel transcription factors may be involved in atherosclerosis, as is suggested by the presence of activated NF-κB in human atherosclerotic lesions. The aim of the present study was to investigate the effects of oxidized LDL (oxLDL) on the NF-κB system in human THP-1 monocytic cells as well as adherent monocytes. Our results demonstrate that short-term incubation of these cells with oxLDL activated p50/p65 containing NF-κB dimers and induced the expression of the target gene IL-8. This activation of NF-κB was inhibited by the antioxidant and H2O2scavenger pyrrolidine dithiocarbamate and the proteasome inhibitor PSI. The oxLDL-induced NF-κB activation was accompanied by an initial depletion of IκB-α followed by a slight transient increase in the level of this inhibitor protein. In contrast, long-term treatment with oxLDL prevented the lipopolysaccharide-induced depletion of IκB-α, accompanied by an inhibition of both NF-κB activation and the expression of tumor necrosis factor-α and interleukin-1β genes. These observations provide additional evidence that oxLDL is a potent modulator of gene expression and suggest that (dys)regulation of NF-κB/Rel is likely to play an important role in atherogenesis.
Thrombin Receptor-Mediated Increase of Two Matrix Metalloproteinases, MMP-1 and MMP-3, in Human Endothelial Cells
Authors: Duhamel-Clérin Emmanuelle , Orvain Christophe , Lanza François , Cazenave Jean-Pierre , Klein-Soyer Claudine
Published: June 2011

AbstractMatrix metalloproteinases (MMPs) are responsible for the degradation of extracellular matrix components and are secreted by a variety of cells including human endothelial cells. Because α-thrombin is known to interact with matrix components and has been shown to activate latent MMP-2 in human umbilical vein endothelial cells, we investigated whether human α-thrombin could also regulate other MMPs secreted by the human saphenous vein or mammary artery endothelial cells (EC). After treatment of EC with increasing concentrations of thrombin for different periods of time, a significantly higher gelatinolytic activity of both MMP-1 and MMP-3 was observed in addition to MMP-2 activation. The effect of thrombin was time and dose-dependent, reaching a maximum at 24 hours. After treatment with 5 NIH U/ml thrombin for 24 hours, Western blotting revealed 9.5- and 4.4-fold increases over control values for MMP-3 and MMP-1, respectively. The synthetic thrombin receptor agonist peptide SFLLRNPNDKYEPF fully reproduced the action of thrombin, whereas chemical inactivation of the catalytic site of thrombin abolished its effect on MMP-1 and MMP-3. Thrombin and SFLLRNPNDKYEPF both induced MMP-3 mRNA synthesis but had no significant influence on constitutive MMP-1 mRNA levels. These results demonstrate that thrombin not only activates latent MMP-2 but also modulates MMP-1 and MMP-3 production in EC, this latter effect being mediated by the G-protein-coupled thrombin receptor. Hence, our present data provide evidence to support the suspected role of thrombin in tissue remodeling and angiogenesis.
Effects of Angiotensin II on Cardiac Function and Peripheral Vascular Structure During Compensated Heart Failure in the Rat
Authors: Heeneman Sylvia , Smits Jos F.M. , Leenders Peter J.A. , Schiffers Paul M.H. , Daemen Mat J.A.P.
Published: June 2011

Abstract
The present experiments were designed to test the hypothesis that the activation of the renin-angiotensin system during compensated heart failure may have adverse effects on cardiac function and change the peripheral vascular structure. ANG II (250 ng/kg/min) or saline (0.9% NaCl) were infused in myocardial-infarcted and sham-operated rats. After 2 weeks, cardiac function and peripheral vascular changes were investigated. Results: ANG II infusion reduced baseline cardiac index in sham rats but did not further reduce this index in ANG II-infused MI rats. Total peripheral resistance was similarly increased in ANG II-infused infarcted and sham rats, and also plasma ANG II concentrations were comparable. ANG II elevated systolic blood pressure by approximately 70 mm Hg in sham rats and increased the medial cross-sectional area of the superior mesenteric artery by 33%. However, ANG II infusions in MI rats resulted in only a minor increase in blood pressure, whereas the cross-sectional area of the superior mesenteric artery did not change. ANG II infusion had no effect on vessel dimensions of the resistance arteries of the pulmonary and mesenteric vascular bed of either group. Calculated ED
50
and peak pressor response to acute ANG II injections were comparable in all groups, confirming the presence of functionally intact AT
1
receptors. The increases in plasma atrial natriuretic peptide (ANP) and nitric oxide (NO) synthase activity (estimated by aortic cyclic GMP concentrations) were higher in ANG II-infused MI rats than in ANG II-infused sham rats. Conclusion: ANG II infusion in rats with and without MI has comparable negative effects on cardiac function but has different effects on blood pressure and vascular structure. The concomitant increases in plasma ANP and NO synthase activity in ANG II-infused MI rats suggest that the growth stimulatory and hypertensive actions of ANG II in sham rats may be counter-regulated by activation of inhibitory neurohumoral systems such as ANP or NO in MI rats.
Effects of Genotype and Diet on Cholesterol Efflux into Plasma and Lipoproteins of Normal, Apolipoprotein A-I-, and Apolipoprotein E-Deficient Mice
Authors: Huang Yadong , Zhu Yanhong , Langer Claus , Raabe Martin , Wu Shili , Wiesenhütter Bernd , Seedorf Udo , Maeda Nobuyo , Assmann Gerd , von Eckardstein Arnold
Published: June 2011

Abstract
We investigated the contribution of apoE to cholesterol efflux into plasmas of normal, apoA-I-, and apoE-deficient mice, which were fed with chow- and cholesterol-rich diets. Plasmas of normal and apoA-I-deficient mice contain apoE in pre-β-migrating VLDL as well as in HDL-like lipoproteins, which have either electrophoretic α- or γ-mobilities. The latter particle resembled γ-LpE in human plasma also by its mobility on nondenaturing two-dimensional electrophoresis. No apoE-containing lipoproteins were found in plasmas of apoE-deficient mice. When apoA-I- and apoE-deficient mice received both chow- and fat-rich diets, their plasmas released significantly less
3
H-cholesterol from radiolabeled fibroblasts than did plasma of normal mice. Removal of apoE from plasmas of normal and apoA-I-deficient mice by anti-apoE immunoaffinity chromatography decreased their cholesterol efflux capacities (per 1 minute/per 1 hour) by 26%/40% (
P
=0.0092/0.0007) and 30%/26% (
P
=0.0092/0.0003), respectively. Net cholesterol efflux from fibroblasts into apoA-I-deficient plasma was 45% lower compared with plasma of normal mice. Incubation of fibroblasts with apoE-deficient plasma caused net influx of cholesterol. Prior addition of human apoE to or removal of apoB-containing lipoproteins from apoE-deficient plasma restored its ability to cause net cholesterol efflux to 50% of normal plasma. Some of the differences between cholesterol efflux into normal and apoE-deficient plasmas were attributable to the failure of apoE-deficient plasmas to take up cell-derived
3
H-cholesterol into γ-LpE. Compared with normal plasma, both apoA-I-deficient and apoE-deficient plasmas were significantly decreased in their activity to esterify cell-derived
3
H-cholesterol. Anti-apoE chromatography decreased significantly cholesterol esterification in normal plasma and apoA-I-deficient plasma but not in apoE-deficient plasma. Taken together, the data provide evidence that apoE is an important contributor to reverse cholesterol transport, partially because of initial uptake of cell-derived cholesterol by γ-LpE and partially because of the contribution of apoE-containing lipoproteins to esterification of cholesterol in plasma.
Lipoprotein(a) Isoforms Display Differences in Affinity for Plasminogen-Like Binding to Human Mononuclear Cells
Authors: Kang C. , Durlach V. , Soulat T. , Fournier C. , Anglés-Cano E.
Published: June 2011

Abstract
Binding of lipoprotein(a) (Lp(a)) to membrane proteins of the monocyte-macrophage cell lineage may be an important event in atheroma formation. Since Lp(a) with distinct apolipoprotein(a) (apo(a)) isoforms may show differences in their affinity with regard to fibrin binding, the existence of such a functional behavior in the interaction of apo(a) in Lp(a) with these cells was explored using the monocytic cell line THP-1. Lp(a) preparations containing small size apo(a) isoforms (
M
r
=450 000 to 550 000) and high molecular mass isoforms (
M
r
≥700 000) were purified from plasmas containing >0.35 g/L of Lp(a) obtained from subjects (n=14) with cardiovascular atherosclerotic disease. Binding of plasminogen to THP-1 cells was performed using the method of radioisotopic dilution. For binding of Lp(a) to cells, the THP-1 monocytic cells were incubated with varying concentrations of the different Lp(a) preparations; cells were then washed and the amount of Lp(a) bound was detected with a radiolabeled polyclonal antibody directed against apo(a). Binding due to kringle interactions with lysine residues was calculated by subtracting from the total bound the amount of Lp(a) bound (≈10%) in the presence of 6-aminohexanoic acid. Analysis of data with the Langmuir equation indicated identical and independent (noninteracting) sites and allowed evaluation of the
K
d
. Binding isotherms of small size isoforms showed saturation and a high affinity (
K
d
=25.8±19 nmol/L) relative to that of plasminogen (
K
d
=1750±760 nmol/L). A similar difference (
K
d
=17.5±7.9 nmol/L versus
K
d
=600±220 nmol/L) was found when binding experiments were performed with a fibrin surface. In contrast, binding isotherms of the high molecular mass isoforms did not show saturation at the highest Lp(a) concentrations used, thus indicating a lower affinity. In conclusion, these results show that apo(a) isoforms may display polymorphism-linked functional heterogeneity with regard to cell binding, which may explain the higher association with cardiovascular risk of small size isoforms. These qualitative differences in the binding of apo(a) isoforms to fibrin or cells may modulate the cardiovascular risk associated with high levels of Lp(a).
Monounsaturated and Polyunsaturated n-6 Fatty Acid–Enriched Diets Modify LDL Oxidation and Decrease Human Coronary Smooth Muscle Cell DNA Synthesis
Authors: Mata Pedro , Varela Odabella , Alonso Rodrigo , Lahoz Carlos , de Oya Manuel , Badimon Lina
Published: June 2011

Abstract
Proliferation of smooth muscle cells (SMCs) plays an important role in atherosclerotic lesion progression. The purpose of this investigation was to examine the effect of diets differing in fatty acid composition on human coronary SMC entry in the cell proliferation cycle. Twenty-four healthy men and women were placed on four consecutive diets lasting 5 weeks each: (1) saturated fatty acid (SFA)–rich diet with palm oil; (2) monounsaturated fatty acid (MUFA)–rich diet with olive oil; (3) polyunsaturated fatty acid (PUFA) n-6–rich diet with sunflower oil; and (4) PUFA n-3–rich diet (3.8 g/d). All diets supplied 35% of calories as fat. Compared with the SFA diet, all unsaturated diets reduced LDL cholesterol. Resistance of LDL to oxidative modification was significantly increased during the MUFA period (
P
<.05). Human coronary SMCs were cultured and induced by sera derived from the different groups.
3
H-Thymidine incorporation into doubling DNA was significantly (
P
<.01) reduced during the MUFA and PUFA n-6 periods but not during the PUFA n-3 diet with respect to the SFA diet. This effect was more pronounced in women than in men. In conclusion, the MUFA-enriched diet reduced SMC DNA synthesis and LDL levels and protected LDL from oxidation. Therefore, these combined effects suggest that an oleic acid–rich Mediterranean diet could be better than PUFA (n-6)– or PUFA (n-3)–rich diets in the prevention of atherosclerosis.
Sustained Anti-CD4/CD8 Treatment Blocks Inflammatory Activation and Intimal Thickening in Mouse Heart Allografts
Authors: Räisänen-Sokolowski Anne , Glysing-Jensen Troels , Mottram Patricia L. , Russell Mary E
Published: June 2011

AbstractWe evaluated inflammatory activation and vascular thickening in a heterotopic murine heart transplant model. C57BL/6J recipient mice received anti-CD4 therapy (days 1 to 4 after transplantation) or sustained, combined anti-CD4/CD8 therapy (days 1 to 4, weekly thereafter). Morphometric analysis of grafts (>95 days) found the mean percentage of vessel occlusion to be 51.7% in allografts treated with anti-CD4, 8.3% in allografts treated with sustained anti-CD4/CD8, and 6.7% in isografts. Mean transcript levels of the adhesion molecules P-selectin, intercellular adhesion molecule 1 (ICAM-1), and leukocyte function-associated antigen 1 (LFA-1) and the cytokines interleukin 4 (IL-4), interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), allograft inflammatory factor 1 (AIF-1), and monocyte chemoattractant protein 1 (MCP-1) were measured with reverse transcription–polymerase chain reaction [RT-PCR] assays using deoxycytidine triphosphate radiolabeled with phosphorus 32 [32P-dCTP]. The assays were normalized against glyceraldehyde-3-phosphate dehydrogenase [G3PDH] Levels were found to be significantly higher in the anti-CD4 group than in the anti-CD4/CD8 group. A strong correlation was also found between the percentage of luminal occlusion and the expression of these markers of inflammation (r=.92-.99,P<.0001). Sustained therapy involving proximal blockade of CD4 and CD8 interrupts pathways leading to inflammation and vascular thickening. However, long-term heart allografts in mice treated with a short course of anti-CD4 display an ongoing inflammatory cell activation that culminates in arteriosclerosis. This model may help examine the role of targeted immune factors using knockout mice to identify those causally involved in vessel thickening.
Topographical Association Between the Cyclin-Dependent Kinases Inhibitor P21, p53 Accumulation, and Cellular Proliferation in Human Atherosclerotic Tissue
Authors: Ihling Christian , Menzel Grit , Wellens Eckhard , Mönting Jürgen Schulte , Schaefer Hans E. , Zeiher Andreas M.
Published: June 2011

Abstract
The cell cycle is controlled by cyclin-dependent protein kinases (CDKs). The activity of these enzymes is directed by inhibitors of CDKs. The 21-kD protein product (P21) of the
WAF1/CIP1
gene, which can be transactivated by the protein product of the tumor suppressor gene
p53
, acts as an inhibitor of cyclin-dependent kinases. To assess whether both P21 and p53 may play a role in the control of cellular proliferation in atherosclerotic lesions, the topographical association between p53, P21, and the proliferation marker MIB1/Ki-67, was analyzed by immunohistochemistry in human carotid atheromatous plaques of 26 patients. p53 immunoreactivity (IR) was present in 26 of 26 cases in the nuclei of virtually all cell types (macrophages [MPs], smooth muscle cells [SMCs], endothelial cells [ECs]) in areas with chronic inflammation in 71.08±8.28% of the nuclei. p53 staining in the control tissue from human coronary arteries was present in 0.3±0.45% of the cells (
P
<.002). P21-IR was present in 24 of 26 specimens in 64.38±10.13% of the cells (controls: 3.8±1.85%,
P
<.002) and localized to nuclei of MPs (CD68 positive) and SMCs (α-actin positive), as well as ECs of microvessels present in 21 specimens (21 of 21) and luminal ECs present in 18 specimens (16 of 18). As shown by double labeling, P21-IR colocalized with p53-IR in most MPs (24 of 24), intimal SMCs (22 of 24), ECs of microvessels (19 of 21), and luminal ECs (10 of 16). Interestingly, few p53-positive cells did not show simultaneous P21-IR, and, conversely, not all P21-positive cells demonstrated p53-IR. MIB1/Ki-67-positive cells were identified in 21 of 26 tissue specimens in 3.53±1.79% of the nuclei (controls: 0%,
P
<.002) and localized principally to MPs bordering the atheromatous lipid core (21 of 26) and to a few scattered SMCs (16 of 26), ECs of microvessels (13 of 21), and luminal ECs (2 of 18). Most importantly, none of the cells coexpressing P21 and p53 were positive for MIB1/Ki-67-IR, indicating the absence of proliferating activity. In summary, this study demonstrates that P21-IR is present in the atherosclerotic plaque and colocalizes with p53 in most MPs, SMCs, and ECs. The lack of proliferation markers in cells coexpressing p53 and P21 suggests that transcriptional activation of the
WAF1/CIP1
gene by p53 may be involved in the control of cellular proliferation in advanced human atherosclerotic plaques.
Effect of Streptozotocin-Induced Hyperglycemia on Lipid Profiles, Formation of Advanced Glycation Endproducts in Lesions, and Extent of Atherosclerosis in LDL Receptor-Deficient Mice
Authors: Reaven Peter , Merat Shiva , Casanada Florencia , Sutphin Mary , Palinski Wulf
Published: June 2011

Abstract
Investigations into the mechanisms by which diabetes accelerates atherosclerosis have been hampered by the lack of suitable animal models. We hypothesized that streptozotocin-treated LDL receptor-deficient mice would be a good model of diabetic atherosclerosis because streptozotocin causes diabetes in the parent C57BL/6 strain and because in these mice diet-induced hypercholesterolemia leads to the formation of advanced atherosclerotic lesions throughout the aorta. Diabetes was induced in 18 mice by intraperitoneal injection of streptozotocin. Low-dose insulin was given subcutaneously to prevent excessive mortality and extreme elevations in triglyceride levels. The control group was subjected to sham injections. Both groups were fed a diet containing .075% cholesterol for six months. Average blood glucose was higher in the diabetic group than in the control group (257±67 mg/dL versus 111±7 mg/dL,
P
<0.05). Although plasma cholesterol was similar (966±399 versus 1002±180 mg/dL) in both groups, VLDL cholesterol was higher whereas LDL cholesterol was lower in the diabetic group. Immunocytochemical analysis demonstrated significantly more advanced glycation endproduct (AGE) epitopes in the artery wall of the diabetic group, whereas staining for oxidation-specific epitopes was similar in both groups. Sera of diabetic mice also contained significantly more IgG autoantibodies that bound to several AGE epitopes than did sera from control mice. Despite the presence of hyperglycemia, diabetic dyslipidemia, and enhanced AGE formation in the diabetic mice, both groups had a similar extent of atherosclerosis (diabetic, 17.3±5.2; control, 16.5±6.6% of the aortic surface). These data suggest that, at least under conditions of marked hypercholesterolemia, hyperglycemia and enhanced AGE formation do not contribute significantly to atherogenesis in LDL
−/−
mice.
Apoptosis After Stent Implantation Compared With Balloon Angioplasty in Rabbits
Authors: Kollum Marc , Kaiser Simone , Kinscherf Ralf , Metz Jürgen , Kübler Wolfgang , Hehrlein Christoph
Published: June 2011

Abstract
Both cell proliferation and apoptosis (programmed cell death) are supposed to play a role in restenosis after angioplasty. We studied these processes in smooth muscle cells (SMCs) and macrophages 1, 4, and 12 weeks after balloon angioplasty or Palmaz-Schatz stent implantation in rabbit iliac arteries. Proliferating cells were visualized by immunostaining with antibodies directed against proliferating cell nuclear antigen. Apoptotic cells were detected using the TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling) technique, propidium iodide staining, and transmission electron microscopy. At all time points, the neointimal cross-sectional area of the arteries was twofold to fourfold greater after stent implantation than after balloon angioplasty. The total number of neointimal cells was similar 1 and 12 weeks after both interventions. The neointimal cell density, however, decreased by 58% between the 1st and the 12th week after stent implantation compared with a 20% decrease after balloon angioplasty (
P
<.01). Stent implantation induced more cell proliferation but also more apoptosis in the media than balloon angioplasty after 1 and 4 weeks. In addition, stent implantation caused more macrophage accumulation and apoptosis in the neointima, but cell proliferation rates did not differ significantly in comparison with balloon angioplasty. The higher rate of apoptosis in the neointima 1 week after stent implantation compared with balloon angioplasty is due to an increased rate of SMC and macrophage death. Macrophage accumulation and apoptosis in the early phase after stent implantation appear to play a role in extracellular matrix secretion, which increases neointima formation after 4 and 12 weeks compared with balloon angioplasty in this model.
Does Insulin Resistance Unite the Separate Components of the Insulin Resistance Syndrome?
Authors: Donahue Richard P. , Bean Judy A. , Donahue Rosemary DeCarlo , Goldberg Ronald B. , Prineas Ronald J.
Published: June 2011

Abstract
A number of coronary heart disease risk factors have been identified that often cluster together to increase the risk of macrovascular disease. This cluster is referred to as the insulin resistance syndrome, and the risk factors commonly include dyslipidemia, elevated blood pressure, an android pattern of body fat distribution, and glucose intolerance. Whether hyperinsulinemia or insulin resistance per se provides a common pathway for these metabolic abnormalities is unclear. The authors studied 50 nondiabetic persons who had completed a euglycemic hyperinsulinemic clamp protocol in addition to a 75-g oral glucose tolerance test and other measures of the coronary risk profile. Using principal-component analysis, we reduced nine coronary risk factors to two uncorrelated factors that explained 54.5% of the variance. Factor 1 consisted of positive loadings for uric acid, systolic and diastolic blood pressure, triglyceride concentration, and waist girth and negative loadings for HDL cholesterol and the rate of insulin-mediated glucose disposal (M, in milligrams per kilogram of body weight per minute). M also loaded on factor 2, along with fasting insulin and glucose concentrations, diastolic blood pressure, and waist girth. The observation that M loaded on both factors suggests that a resistance to insulin action may provide the mechanism uniting the features of the insulin resistance syndrome. Hyperinsulinemia with concomitant insulin resistance may be necessary to produce this metabolic derangement, as well as the increased risk of macrovascular complications.