bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary Antioxidants Do Not Reduce Fatty Streak Formation in the C57BL/6 Mouse Atherosclerosis Model
Authors: Munday John S. , Thompson Keith G. , James Kerry A. C. , Manktelow B. William
Published: June 2011

Abstract
—Epidemiological studies and animal trials have suggested that dietary antioxidants protect against atherosclerosis. To test this hypothesis, C57BL/6 mice were fed atherogenic diets supplemented with either vitamin E or butylated hydroxytoluene (BHT). Three groups of 20 mice were fed for 15 weeks on diets containing 1% cholesterol and 0.5% cholic acid. The diet of two groups was supplemented with either 2% vitamin E or 1% BHT. The control group received no antioxidant supplements. The lowest mean serum cholesterol concentration was measured in mice supplemented with vitamin E. Mean serum HDL cholesterol concentrations were highest in the control group, which also had the highest ratio of HDL cholesterol to total cholesterol. Mice fed BHT developed a significantly greater area of aortic fatty streak lesions than the other two groups. However, despite having a more atherogenic lipoprotein profile, mice fed vitamin E developed a level of fatty streak formation similar to the control group. At the end of the trial, mice consuming the vitamin E- and BHT-supplemented diets had higher serum total antioxidant levels than the control mice. Because of changes to lipid metabolism caused by both vitamin E and BHT, the results of this study cannot be used to support the hypothesis that antioxidants confer protection against atherosclerosis. The results do, however, raise the possibility that other studies demonstrating an antiatherogenic action of vitamin E and BHT may have been influenced by their effects on lipid metabolism.
Hyperhomocysteinemia Is Associated With an Increased Risk of Cardiovascular Disease, Especially in Non–Insulin-Dependent Diabetes Mellitus
Authors: Hoogeveen Ellen K. , Kostense Pieter J. , Beks Pieter J. , Mackaay Albert J. C. , Jakobs Cornelis , Bouter Lex M. , Heine Robert J. , Stehouwer Coen D. A.
Published: June 2011

Abstract
—A high serum total homocysteine (tHcy) level is an independent risk factor for cardiovascular disease. Because it is not known whether the strength of the association between hyperhomocysteinemia and cardiovascular disease is similar for peripheral arterial, coronary artery, and cerebrovascular disease, we compared the three separate risk estimates in an age-, sex-, and glucose tolerance–stratified random sample (n=631) from a 50- to 75-year-old general white population. Furthermore, we investigated the combined effect of hyperhomocysteinemia and diabetes mellitus with regard to cardiovascular disease. The prevalence of fasting hyperhomocysteinemia (>14.0 μmol/L) was 25.8%. After adjustment for age, sex, hypertension, hypercholesterolemia, diabetes, and smoking, the odds ratios (ORs; 95% confidence intervals) per 5 -μmol/L increment in tHcy were 1.44 (1.10 to 1.87) for peripheral arterial, 1.25 (1.03 to 1.51) for coronary artery, 1.24 (0.97 to 1.58) for cerebrovascular, and 1.39 (1.15 to 1.68) for any cardiovascular disease. After stratification by glucose tolerance category and adjustment for the classic risk factors and serum creatinine, the ORs per 5 -μmol/L increment in tHcy for any cardiovascular disease were 1.38 (1.03 to 1.85) in normal glucose tolerance, 1.55 (1.01 to 2.38) in impaired glucose tolerance, and 2.33 (1.11 to 4.90) in non–insulin-dependent diabetes mellitus (
P
=.07 for interaction). We conclude that the magnitude of the association between hyperhomocysteinemia and cardiovascular disease is similar for peripheral arterial, coronary artery, and cerebrovascular disease in a 50- to 75-year-old general population. High serum tHcy may be a stronger (1.6-fold) risk factor for cardiovascular disease in subjects with non–insulin-dependent diabetes mellitus than in nondiabetic subjects.
Cytokines Stimulate GTP Cyclohydrolase I Gene Expression in Cultured Human Umbilical Vein Endothelial Cells
Authors: Katusic Zvonimir S. , Stelter Adele , Milstien Sheldon
Published: June 2011

Abstract
—In vascular endothelial cells, tetrahydrobiopterin serves as an essential cofactor required for enzymatic activity of nitric oxide synthase. GTP cyclohydrolase I is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Previous studies have demonstrated that proinflammatory cytokines stimulate production of tetrahydrobiopterin in endothelial cells. Long-term regulation of GTP cyclohydrolase I gene expression in endothelium has not been studied. The present study was designed to determine whether the cytokines tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), and interleukin-1β (IL-1β) stimulate tetrahydrobiopterin synthesis by increasing expression of GTP cyclohydrolase I mRNA in endothelial cells. The relative reverse transcription polymerase chain reaction was used to quantify expression of GTP cyclohydrolase I mRNA in cultured human umbilical vein endothelial cells. Nuclear run-on assay was performed to determine the transcription rate of GTP cyclohydrolase I gene. GTP cyclohydrolase I enzymatic activity and production of tetrahydrobiopterin were measured in cell extracts. After incubation with TNF-α (2 μg/mL), INF-γ (200 U/mL), and IL-1β (5 U/mL) for 24 hours, significantly increased expression of GTP cyclohydrolase I mRNA was detected. Cytokines increased the transcription rate of GTP cyclohydrolase I 3.6-fold. This increase was associated with increased GTP cyclohydrolase I enzymatic activity and elevation of intracellular levels of tetrahydrobiopterin. An RNA synthesis inhibitor, actinomycin D (2 μg/mL), inhibited cytokine-induced expression of GTP cyclohydrolase I gene. A protein synthesis inhibitor, cycloheximide (0.5 μg/mL), did not affect expression of GTP cyclohydrolase I mRNA but blocked the increase in enzyme activity, as well as production of tetrahydrobiopterin. Incubation of endothelial cells for 24 hours in the presence of 8-bromoadenosine 3′:5′-cyclic monophosphate (10
−3
mol/L) did not affect expression of GTP cyclohydrolase I mRNA. These results demonstrate that in vascular endothelial cells, cytokines increase production of tetrahydrobiopterin by stimulating expression of GTP cyclohydrolase I gene. This effect is apparently due to increased transcription rather than stabilization of mRNA. Regulation of GTP cyclohydrolase I gene expression by cytokines may play an important role in control of endothelial nitric oxide synthesis.
Association of Vitamin K–Dependent Coagulation Proteins and C4b Binding Protein With Triglyceride-Rich Lipoproteins of Human Plasma
Authors: Xu Ning , Dahlbäck Björn , Öhlin Ann-Kristin , Nilsson Åke
Published: June 2011

Abstract
—The triglyceride (TG) concentration in plasma is an independent risk factor for coronary heart disease. There is evidence that TG-rich lipoprotein (TGRLP), ie, chylomicrons (CMs), chylomicron remnants (CMRs), and VLDLs associate with factor VII and prothrombin and that the association enhances a platelet factor Xa–mediated prothrombin activation when the CM-prothrombin complex is exposed to platelets. In this study, we examined the association of the vitamin K–dependent coagulation factors VII, IX, X, and prothrombin, as well as the anticoagulation protein C and its cofactor protein S, in plasma lipoproteins obtained from human fasting and postprandial plasma. We also analyzed some other proteins that are related to the coagulation system but not to vitamin K–dependent proteins, including factor V, serum amyloid P component (SAP), C4b binding protein (C4BP), and thrombomodulin (TM), and as a control, Ig G. Human TGRLP (
d
<1.006 kg/L), LDL (
d
=1.006 to 1.063 kg/L), and HDL (
d
=1.063 to 1.210 kg/L) were separated from normal subjects both in fasting and 2 to 3 hours after the ingestion of a meal containing 100 g fat. The different coagulation proteins, SAP, C4BP, TM, and Ig G were determined by SDS-polyacrylamide gel electrophoresis combined with Western blotting, using specific polyclonal or monoclonal antibodies, and were visualized by peroxidase staining. All the vitamin K–dependent proteins associate with TGRLP in both fasting and postprandial plasma, but not with LDL or HDL. Factor V, SAP, TM, and Ig G were not found in any lipoprotein classes. C4BP, which is a regulatory protein of the classic pathway of the complement system and which binds protein S in vivo to regulate blood coagulation, was present in TGRLP, especially postprandial, but not in LDL or HDL. The amounts of prothrombin, protein S, and C4BP in postprandial TGRLP were larger than those in fasting TGRLP. Vitamin K–dependent procoagulation and anticoagulation proteins, as well as C4BP, could be associated with TGRLP in vivo. If the association enhances prothrombin activation, this effect may thus be counteracted by simultaneous binding of protein S.
Quantification and Characterization of Human Endothelial Cell–Derived Tissue Factor Pathway Inhibitor-2
Authors: Iino Masaki , Foster Donald C. , Kisiel Walter
Published: June 2011

Abstract
—Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a serine protease inhibitor consisting of three tandemly-arranged Kunitz-type protease inhibitor domains. While TFPI-2 is a potent inhibitor of trypsin, plasmin, kallikrein, and factor XIa in the test tube, the function of this inhibitor in vivo remains unclear. In the present study, we investigated the synthesis and secretion of TFPI-2 by cultured endothelial cells derived from human umbilical vein, aorta, saphenous vein, and dermal microvessels to gain insight into its biological function. While all endothelial cells examined synthesized and secreted TFPI-2, dermal microvascular endothelial cells synthesized threefold to sevenfold higher levels of TFPI-2. Approximately 60% to 90% of the TFPI-2 secreted by endothelial cells was directed to the subendothelial extracellular matrix (ECM). When cultured human umbilical vein endothelial cells were stimulated with inflammatory mediators such as phorbol 12-myristate,13-acetate; endotoxin; and tumor necrosis factor-α, TFPI-2 synthesis by these cells increased twofold to 14-fold. Recombinant TFPI-2 bound to dermal microvascular endothelial cell monolayers and its ECM in a specific, dose-dependent, and saturable manner with K
d
values of 21 and 24 nmol/L, respectively. TFPI-2 interacted with 4.5×10
10
sites/cm
2
(3×10
5
sites/cell) and 2.3×10
11
sites/cm
2
on endothelial cells and ECM, respectively. In the presence of rabbit anti–TFPI-2 IgG, but not preimmune IgG, endothelial cells dissociated from the culture flask in a time- and IgG concentration–dependent manner. Our findings provide evidence that endothelial cell–derived TFPI-2 is primarily secreted into the abluminal space and presumably plays an important role in maintaining the integrity of the ECM essential for cell attachment.
Oral Contraceptives and Hormone Replacement Therapy Do Not Increase the Incidence of Arterial Thrombosis in a Nonhuman Primate Model
Authors: Bellinger Dwight A. , Williams J. Koudy , Adams Michael R. , Honoré Erika K. , Bender Diane E.
Published: June 2011

Abstract
—Older oral contraceptive (OC) formulations containing high doses of potent synthetic estrogens and progestins are associated with increased risk of thrombosis. To examine the effects of current low-dose OC and hormone replacement therapy (HRT) regimens on arterial thrombosis, premenopausal and surgically postmenopausal cynomolgus monkeys were divided into four treatment groups. Premenopausal monkeys were given either no OCs or ethinyl estradiol and levonorgestrel as an OC at a dose equivalent to that currently given to women. Postmenopausal monkeys were given either no HRT or conjugated equine estrogens and medroxyprogesterone as an HRT at a dose equivalent to that currently given to women. The monkeys were fed an atherogenic diet containing these treatments for 27 to 30 months. At the end of this time, arterial thrombosis was evaluated with a standardized stenosis/injury procedure in the left carotid artery. Blood flow velocity was monitored for cyclic or permanent occlusive thrombosis. The current OC and HRT regimens did not increase the susceptibility of the artery wall to develop an occlusive thrombus following injury and stenosis. In fact, there was a reduction in the incidence of thrombosis in the OC animals compared with untreated controls. Increased amounts of atherosclerosis were associated with an increased incidence of occlusive arterial thrombosis. Several selected coagulation parameters [von Willebrand factor, protein C, lipoprotein(a), and platelet aggregation] did not appear to be associated with either the amount of atherosclerosis or incidence of arterial thrombosis.
Differential Induction of Cyclooxygenase-2 in Human Arterial and Venous Smooth Muscle
Authors: Bishop-Bailey David , Pepper John R. , Larkin Simon W. , Mitchell Jane A.
Published: June 2011

Abstract
—Two isoforms of cyclooxygenase (COX) have been identified: a constitutive isoform (COX-1), found in abundance in platelets and the vascular endothelium, and an “inflammatory” cytokine-inducible isoform (COX-2). Because COX metabolites regulate vascular smooth muscle cell (SMC) function and the interaction between the vessel and circulating components, we have investigated the possibility that COX-2 can be induced in human arterial or venous SMC. Untreated venous or arterial cells contained undetectable levels of COX-1 or COX-2 and released low levels of metabolites. After stimulation with interleukin-1β, tumor necrosis factor-α, interferon-γ, and bacterial lipopolysaccharide, both venous and arterial SMC expressed COX-2 protein and released increased amounts of prostaglandins. In addition, the induced release of PGE
2
was inhibited by the COX-2–selective inhibitor, L-745,337. When cells were treated with the mixture of cytokines, venous SMC expressed greater amounts of COX-2 protein and released more prostaglandins than arterial SMC. Furthermore, when COX-2 activity was blocked by L-745,337, COX-2 expression in arterial SMC, but not in venous SMC, increased. Thus, this article describes, for the first time, that COX-2 is expressed in greater amounts in venous SMC than in arterial SMC. Moreover, we show that this “differential induction” is due to a negative-feedback pathway for COX-2 expression in arterial SMC but not in venous SMC. The ability of COX-2 activity to limit COX-2 expression in some cells but not others may contribute to the highly developed mechanisms involved in prostanoid release.
Vitamin C Protects Human Arterial Smooth Muscle Cells Against Atherogenic Lipoproteins
Authors: Siow Richard C. M. , Sato Hideyo , Leake David S. , Pearson Jeremy D. , Bannai Shiro , Mann Giovanni E.
Published: June 2011

Abstract
—Glutathione (GSH) plays a key role in cellular antioxidant defenses by scavenging reactive oxygen species and reducing lipid peroxides. Intracellular GSH levels are regulated by transport of its precursor
l
-cystine via system x
c
−
, which can be induced by oxidant stress. As oxidatively modified low density lipoproteins (LDLs) contribute to impaired vascular reactivity and the formation of atherosclerotic lesions, we have examined the effects of oxidized LDL and the antioxidant vitamins C and E on the
l
-cystine–GSH pathway in human umbilical artery smooth muscle cells (HUASMCs). Oxidized LDL, but not native LDL, elevated intracellular GSH levels and
l
-cystine transport via system x
c
−
in a time-dependent (up to 24 hours) and dose-dependent (10 to 100 μg · mL
−1
) manner. These increases were dependent on protein synthesis and the extent of LDL oxidation, but the induction of
l
-cystine transport activity was independent of GSH synthesis. Pretreatment of HUASMCs for 24 hours with vitamin E (100 μmol/L) attenuated oxidized LDL–mediated increases in GSH, whereas pretreatment with vitamin C depressed basal levels and abolished oxidized LDL–induced increases in GSH and
l
-cystine transport in a time-dependent (3 to 24 hours) and dose-dependent (10 to 100 μmol/L) manner. Pretreatment of cells with dehydroascorbate had no effect on oxidized LDL–mediated increases in
l
-cystine transport and only marginally attenuated increases in GSH. Our findings provide the first evidence that vitamin C spares endogenous adaptive antioxidant responses in human vascular smooth muscle cells exposed to atherogenic oxidized LDL.
HMG-CoA Reductase Inhibitors Reduce MMP-9 Secretion by Macrophages
Authors: Bellosta S. , Via D. , Canavesi M. , Pfister P. , Fumagalli R. , Paoletti R. , Bernini F.
Published: June 2011

Abstract
—Macrophages secrete matrix metalloproteinases (MMPs) that may weaken the fibrous cap of atherosclerotic plaque, predisposing its fissuration. The 92-kDa gelatinase B (MMP-9) has been identified in abdominal aortic aneurysms and in atherosclerotic tissues. Fluvastatin, through the inhibition of the isoprenoid pathway, inhibits major processes of atherogenesis in experimental models (smooth muscle cell migration and proliferation and cholesterol accumulation in macrophages). We studied the effect of fluvastatin on the activity of MMP-9 in mouse and human macrophages in culture. Conditioned media of cells treated for 24 hours with fluvastatin were analyzed by gelatin zymography. In mouse macrophages, fluvastatin (5 to 100 μmol/L) significantly inhibited in a dose-dependent manner MMP-9 activity from 20% to 40% versus control. The drug, at a concentration as low as 5 μmol/L, inhibited MMP-9 activity (≈30%) in human monocyte–derived macrophages as well. Phorbol esters (TPA, 50 ng/mL) stimulated MMP-9 activity by 50%, and fluvastatin inhibited this enhanced activity up to 50% in both mouse and human macrophages. The above results on the secretion of MMP-9 were confirmed by Western blotting and ELISA. The inhibitory effect of fluvastatin was overcome by the simultaneous addition of exogenous mevalonate (100 μmol/L), a precursor of isoprenoids. Fluvastatin’s effect was fully reversible, and the drug did not cause any cellular toxicity. The statin did not block directly the in vitro activation of the secreted protease. Similar data were obtained with simvastatin. Altogether, our data indicate an inhibition of MMP-9 secretion by the drug. This effect is mediated by the inhibition of synthesis of mevalonate, a precursor of numerous derivatives essential for several cellular functions.
Unsaturated Fatty Acids Increase Plasminogen Activator Inhibitor-1 Expression in Endothelial Cells
Authors: Nilsson Lennart , Banfi Cristina , Diczfalusy Ulf , Tremoli Elena , Hamsten Anders , Eriksson Per
Published: June 2011

Abstract
—In vivo studies have demonstrated a strong positive correlation between plasma very low density lipoprotein (VLDL) triglyceride and plasma plasminogen activator inhibitor-1 (PAI-1) activity levels. Furthermore, VLDL has been shown to induce PAI-1 secretion from cultured endothelial cells. In contrast, no or variable effects on PAI-1 secretion have been reported for native low density lipoprotein. It could be speculated that fatty acids derived from VLDL triglycerides are the actual mediators, resulting in an enhanced secretion of PAI-1. In the present study, we have analyzed the effects of both saturated and unsaturated fatty acids on PAI-1 expression and secretion by endothelial cells. Addition of 0 to 50 μmol/L of either palmitic acid or stearic acid had no effect on PAI-1 secretion from human umbilical vein endothelial cells or EA.hy926 cells. In contrast, addition of oleic acid, linoleic acid, linolenic acid, and eicosapentaenoic acid resulted in a significant increase in PAI-1 secretion from both cell types. Northern blot analysis of PAI-1 mRNA levels was in agreement with these findings. Transfection experiments demonstrated that addition of linolenic acid and eicosapentaenoic acid significantly increased PAI-1 transcription. The fatty acid response region was localized to a previously described VLDL-inducible region of the PAI-1 promoter. Electromobility shift assays demonstrated that unsaturated fatty acids induced the same complex as did VLDL, whereas saturated fatty acids had no effect. Furthermore, it was demonstrated that the activation procedure did not involve fatty acid oxidation to any significant extent. In conclusion, the present study demonstrates that unsaturated fatty acids increase PAI-1 transcription and secretion by endothelial cells in vitro. The effect appears to be mediated by a previously described VLDL-inducible transcription factor.
Visceral Fat Accumulation Is an Important Determinant of PAI-1 Levels in Young, Nonobese Men and Women
Authors: Giltay E. J. , Elbers J. M. H. , Gooren L. J. G. , Emeis J. J. , Kooistra T. , Asscheman H. , Stehouwer C. D. A.
Published: June 2011

Abstract
—Increased plasminogen activator inhibitor type-1 (PAI-1) levels, leading to impaired fibrinolysis, are associated with increased visceral fat in middle-aged and obese subjects. It is unknown, however, whether this association is independent of other disturbances clustered in the insulin resistance syndrome. We analyzed this association in young, nonobese transsexual men and women before and after administration of cross-sex steroids, which potentially influence many elements of the insulin resistance syndrome, including PAI-1 levels and visceral fat accumulation. We assessed the visceral fat area (by MRI); total body fat; insulin sensitivity (with a glucose clamp technique); and plasma levels of PAI-1, insulin, and triglycerides in young (<37 years old), nonobese (body mass index <28 kg/m
2
), healthy men (n=18) and women (n=15) before and after 12 months of cross-sex hormone administration. Men were treated with ethinyl estradiol 100 μg/d plus cyproterone acetate 100 mg/d, and women were treated with testosterone esters 250 mg IM every 2 weeks. At baseline, only visceral fat area was significantly correlated with plasma PAI-1 levels in both men (
r
=0.57,
P
=0.03) and women (
r
=0.59,
P
=0.03). In multivariate linear regression analysis, this association was independent of total body fat, insulin sensitivity, and plasma levels of triglycerides and insulin. After 12 months of cross-sex hormone administration, the plasma PAI-1 levels were no longer correlated with visceral fat (which had increased).We conclude that in young, nonobese men and women, visceral fat area is an important determinant of plasma PAI-1 levels. After cross-sex hormone administration, this association was no longer demonstrable.
Natriuretic Factors and Nitric Oxide Suppress Plasminogen Activator Inhibitor-1 Expression in Vascular Smooth Muscle Cells
Authors: Bouchie Julie L. , Hansen Hans , Feener Edward P.
Published: June 2011

Abstract
—Increased expression of plasminogen activator inhibitor-1 (PAI-1) has been reported in atherosclerotic and balloon-injured vessels. Little is known regarding the factors and mechanisms that may negatively regulate PAI-1 expression. In this report, the effect of cGMP-coupled vasoactive hormones, including natriuretic factors and nitric oxide, on the regulation of PAI-1 expression in vascular smooth muscle cells was examined. Atrial natriuretic factor 1–28 (ANF) and C-type natriuretic factor-22 (CNP) reduced angiotensin II (Ang II)– and platelet-derived growth factor–stimulated PAI-1 mRNA expression in rat aortic smooth muscle cells by 50% to 70%, with corresponding reductions in PAI-1 protein release. Treatment of human aortic smooth muscle cells with CNP similarly inhibited both platelet-derived growth factor–induced PAI-1 mRNA expression and PAI-1 protein release by 50%. Dose-response studies revealed that the inhibitory effects of CNP and ANF on PAI-1 expression were concentration dependent, with IC
50
s of ≈1 nmol/L for both natriuretic peptides. Ang II–stimulated PAI-1 expression was also inhibited by the nitric oxide donor
S
-nitroso-
N
-acetylpenicillamine. The membrane-permeant cGMP analogue 8-Br-cGMP reduced Ang II–stimulated PAI-1 expression by 60%, and an inhibitor of soluble guanylyl cyclase (1
H
-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) significantly impaired the inhibitory effects of
S
-nitroso-
N
-acetylpenicillamine on Ang II–stimulated PAI-1 expression. Studies of PAI-1 mRNA stability in cells treated with actinomycin D showed that ANF did not alter PAI-1 mRNA half-life, suggesting that natriuretic factors reduce PAI-1 transcription. These data show that natriuretic factors and nitric oxide, via a cGMP-dependent mechanism, inhibit PAI-1 synthesis in vascular smooth muscle cells. Thus, cGMP-coupled vasoactive hormones may play an important role in suppressing vascular PAI-1 expression.
Arterial Injury by Cholesterol Oxidation Products Causes Endothelial Dysfunction and Arterial Wall Cholesterol Accumulation
Authors: Rong James X. , Rangaswamy Shanthini , Shen Lijiang , Dave Ravi , Chang Yi H. , Peterson Hazel , Hodis Howard N. , Chisolm Guy M. , Sevanian Alex
Published: June 2011

Abstract
—Cholesterol oxidation products (ChOx) have been reported to cause acute vascular injury in vivo; however, the pharmacokinetics of ChOx after administration and the mechanisms by which they cause chronic vascular injury are not well understood. To further study the pharmacokinetics and atherogenic properties of ChOx, New Zealand White rabbits were injected intravenously (70 mg per injection, 20 injections per animal) with a ChOx mixture having a composition similar to that found in vivo during a 70-day period. Total ChOx concentrations in plasma peaked almost immediately after a single injection, declined rapidly, and returned to preinjection levels in 2 hours. After multiple injections, the ChOx concentrations rose gradually to levels 2- to 3-fold above baseline levels, increasing mostly in the cholesteryl ester fraction of LDL and VLDL. Rabbit serum and the isolated LDL/VLDL fraction containing elevated ChOx concentrations were cytotoxic to V79 fibroblasts and rabbit aortic endothelial cells. At the time of killing, cholesterol levels in the aortas from ChOx-injected rabbits were significantly elevated despite the fact that plasma cholesterol levels remained in the normal range. In addition, aortas from the ChOx-injected rabbits retained more
125
I-labeled horseradish peroxidase, measured 20 minutes after intravenous injection. Transmural concentration profiles across the arterial wall also showed increased horseradish peroxidase accumulation in the inner half of the media from the thoracic aorta in ChOx-injected rabbits. In conclusion, ChOx injection resulted in accumulation of circulating ChOx and induced increased vascular permeability and accumulation of lipids and macromolecules. This study reveals that even under normocholesterolemic conditions, ChOx can cause endothelial dysfunction, increased macromolecular permeability, and increased cholesterol accumulation, parameters believed to be involved in the development of early atherosclerotic lesions.
Serum Homocysteine and Risk of Coronary Heart Disease and Cerebrovascular Disease in Elderly Men
Authors: Stehouwer Coen D. A. , Weijenberg Matty P. , van den Berg Michiel , Jakobs Cornelis , Feskens Edith J. M. , Kromhout Daan
Published: June 2011

Abstract
—Hyperhomocysteinemia is an independent risk factor for atherosclerotic disease in the middle-aged. We investigated whether a high serum homocysteine level is a risk factor for vascular disease in 878 elderly men (mean age at baseline, 71.5 years; range, 64 to 84 years) in a population-based, representative cohort followed up for 10 years in Zutphen, the Netherlands. Thirty-one percent had nonfasting homocysteine levels ≥17 μmol/L. After adjustment for other major risk factors, high homocysteine levels at baseline (the third compared with the first tertile) were associated with an increased baseline prevalence of myocardial infarction (odds ratio [OR], 1.81; 95% confidence interval [CI], 1.07 to 3.08;
P
for trend, 0.03) and with a marginally significant increase in the risk of dying of coronary heart disease (relative risk [RR], 1.58; 95% CI, 0.93 to 2.69;
P
for trend, 0.09) but not with an increased risk of first-ever myocardial infarction. In addition, high homocysteine levels at baseline were associated with an increased baseline prevalence of stroke (OR, 4.61; 95% CI, 1.79 to 11.89;
P
for trend, 0.002) and with an increased risk of dying of cerebrovascular disease in subjects without hypertension (RR, 6.18; 95% CI, 2.28 to 16.76) but not in those with hypertension. High homocysteine levels were associated with an increased risk of first-ever stroke among normotensive subjects that was not statistically significant (RR, 1.77 [95% CI, 0.83 to 3.75;
P
for trend, 0.14]). In a general population of elderly men, a high homocysteine level is common and is strongly associated with the prevalence of coronary heart disease and cerebrovascular disease. It is a strong predictive factor for fatal cerebrovascular disease in men without hypertension but less so for coronary heart disease.
Plasma Extracellular Superoxide Dismutase Levels in an Australian Population With Coronary Artery Disease
Authors: Wang X. L. , Adachi T. , Sim A. S. , Wilcken D. E. L.
Published: June 2011

Abstract
—In vitro experiments suggest that free radicals may contribute importantly to atherogenesis. Superoxide dismutase (SOD), particularly extracellular SOD (EC-SOD), which accounts for the majority of SOD biological activity, is a major superoxide scavenger. We explored factors that may affect plasma EC-SOD levels measured by ELISA and assessed the association between plasma EC-SOD and coronary artery disease documented angiographically in 590 white Australian patients ≤65 years old. Mean±SEM plasma EC-SOD in female patients (113.6±13.2 ng/mL) was significantly higher than in male patients (86.6±5.1 ng/mL,
P
<0.0001), and all 19 patients with levels >400 ng/mL were heterozygous for the Arg213→Gly mutation at the EC-SOD gene; there was also a positive correlation with age (
r
=0.131,
P
=0.0016). Plasma EC-SOD in current smokers (75.0±9.3 ng/mL) was much lower than in nonsmokers (111.7±8.2 ng/mL,
P
<0.01), and ex-smokers had intermediate levels (84.3±7.1 ng/mL). Levels were significantly lower in patients with than in those without a history of acute myocardial infarction (MI) (76.1±7.5 versus 110.1±6.0 ng/mL,
P
<0.05), and low plasma EC-SOD was independently associated with an increased likelihood of a history of MI (OR, 2.04; 95% CI, 1.10 to 3.82); higher EC-SOD levels also tended to be associated with delayed onset of MI. In conclusion, our study establishes that in patients assessed by coronary angiography, circulating EC-SOD is lower in men than in women and in smokers of each sex and that low levels are independently associated with a history of MI. These findings are consistent with EC-SOD’s being protective and contributing to reduced coronary risk.
Chemokine Receptor CCR2 Expression and Monocyte Chemoattractant Protein-1–Mediated Chemotaxis in Human Monocytes
Authors: Han Ki Hoon , Tangirala Rajendra K. , Green Simone R. , Quehenberger Oswald
Published: June 2011

Abstract—The subendothelial accumulation of macrophage-derived foam cells is one of the hallmarks of atherosclerosis. The recruitment of monocytes to the intima requires the interaction of locally produced chemokines with specific cell surface receptors, including the receptor (CCR2) for monocyte chemoattractant protein-1 (MCP-1). We have previously reported that monocyte CCR2 gene expression and function are effectively downregulated by proinflammatory cytokines. In this study we identified low density lipoprotein (LDL) as a positive regulator of CCR2 expression. Monocyte CCR2 expression was dramatically increased in hypercholesterolemic patients compared with normocholesterolemic controls. Similarly, incubation of human THP-1 monocytes with LDL induced a rapid increase in CCR2 mRNA and protein. By 24 hours the number of cell surface receptors was doubled, causing a 3-fold increase in the chemotactic response to MCP-1. The increase in CCR2 expression and chemotaxis was promoted by native LDL but not by oxidized LDL. Oxidized LDL rapidly downregulated CCR2 expression, whereas reductively methylated LDL, which does not bind to the LDL receptor, had only modest effects on CCR2 expression. A neutralizing anti–LDL receptor antibody prevented the effect of LDL, suggesting that binding and internalization of LDL were essential for CCR2 upregulation. The induction of CCR2 expression appeared to be mediated by LDL-derived cholesterol, because cells treated with free cholesterol also showed increased CCR2 expression. These data suggest that elevated plasma LDL levels in conditions such as hypercholesterolemia enhance monocyte CCR2 expression and chemotactic response and potentially contribute to increased monocyte recruitment to the vessel wall in chronic inflammation and atherogenesis.
Vitronectin Expression and Interaction With Receptors in Smooth Muscle Cells From Human Atheromatous Plaque
Authors: Dufourcq Pascale , Louis Huguette , Moreau Catherine , Daret Danièle , Boisseau Michel R. , Lamazière Jean Marie Daniel , Bonnet Jacques
Published: June 2011

Abstract
—Vitronectin (VN) is a plasma glycoprotein that promotes cell attachment and induces migration of human smooth muscle cells (SMCs) in culture. VN has been observed to accumulate in human atherosclerotic plaques, although its origin and role in atherosclerosis are not yet established. In the present experiments, synthesis of VN by intimal cells and its colocalization with receptors, αvβ3 and αvβ5, were studied by in situ hybridization and immunohistochemistry on 15 human atherosclerotic plaques from carotid arteries obtained after surgery. Strong VN protein and mRNA expression was observed in the intima and in the media. In the intima, VN mRNA expression was colocalized with SMCs, indicating that these cells produce VN, which may account for its accumulation in atherosclerotic plaques. In SMCs in culture, immunoprecipitation after metabolic labeling demonstrated that human SMCs do synthesize vitronectin. Confocal microscopic examination showed that VN colocalized with its receptors, αvβ3 and αvβ5, in the atherosclerotic intima. However, the distribution of the VN receptors on SMCs in culture in contact with VN was different. These observations suggest that VN plays various parts in atherogenesis via different SMC membrane receptors. (

Arterioscler Thromb Vasc Biol. 1998;18:168-176.)
Angiotensin I-Converting Enzyme Genotype Influences Arterial Response to Injury in Normotensive Rats
Authors: Challah Mireille , Villard Eric , Philippe Monique , Ribadeau-Dumas Antoine , Giraudeau Bruno , Janiak Philip , Vilaine Jean-Paul , Soubrier Florent , Michel Jean-Baptiste
Published: June 2011

Abstract
—Two normotensive strains of rat, the Lou and Brown Norway (BN) strains, have contrasting levels of plasma angiotensin-converting enzyme (ACE). To investigate the degree of genetic determination of ACE expression, a polymorphic marker of the ACE gene was analyzed in inbred rats of the two strains. The two inbred strains were shown to bear different alleles for a polymorphic marker at the ACE gene. The segregation of the alleles of this marker and the plasma ACE levels were studied in a group of F2 rats issued from a cross between Lou and BN rats. The degree of genetic determination of plasma ACE activity was estimated to be 94% in the F2 cohort. The ACE locus accounts for 74% of total plasma ACE variance. ACE activity and mRNA expression in lungs were also genetically determined. The difference observed in ACE mRNA accumulation in the lungs between the two strains was due to a difference in the transcriptional rate of the ACE gene, as shown in nuclear run-on experiments. No differences were observed in arterial blood pressure of homozygous F2 progeny. In these animals, ACE genotype did not interfere with the pressor or the depressor responses to ACE-dependent vasoactive peptides. There was a significant effect of strain on constitutive or inducible membrane or soluble ACE activity in primary cultures of vascular cells. Neointima formation in the carotid artery 14 days after balloon injury was also influenced by the genotype in F2 homozygous progeny, whereas the medial area was not. These results demonstrate that there is a close relationship between the genetically determined ACE expression and the inducibility of the ACE gene. The degree of genetic determination of ACE expression in inbred rat strains offers a unique opportunity to study the interaction between genetic and environmental determinants of ACE expression and its involvement in response to experimental cardiovascular and renal injury.
Prolonged Activation of Prothrombin on the Vascular Wall After Arterial Injury
Authors: Ghigliotti Giorgio , Waissbluth Alvaro R. , Speidel Christopher , Abendschein Dana R. , Eisenberg Paul R.
Published: June 2011

Abstract
—This study was designed to characterize the relative roles of bound Xa/Va and thrombin activity in vascular wall procoagulant activity after balloon-induced injury and the extent to which intravenous aspirin and heparin attenuate procoagulant activity associated with the vascular wall. Abdominal aortic injury was induced in rabbits by overinflation and multiple passages of a 4F embolectomy catheter. Rabbits were killed 15 minutes or 4, 8, 24, 48, 72, 96, or 120 hours after injury. Aortic segments were incubated ex vivo to define bound procoagulant activity. Thrombin activity bound to the aorta was detected by 4 hours after injury and was most marked over the first 24 hours, as estimated by increases in concentration of fibrinopeptide A during incubation of segments with recalcified barium-adsorbed plasma or activity against the thrombin-synthetic substrate S-2238. Based on comparison with purified human thrombin incubated under the same conditions, a maximum of 0.04 to 0.1 nmol/L per square centimeter of thrombin activity was associated with the vascular wall during the first 24 hours and remained detectable for 72 hours. In contrast, bound Xa/Va complex activity to injured segments was detected within 15 minutes and induced activation of prothrombin added to recalcified barium-adsorbed plasma incubated with injured segments for 96 hours. Aspirin (15 mg/kg) administered 30 minutes before injury attenuated
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In-platelet deposition at 4 hours by 67%, with an associated decrease in bound Xa/Va and thrombin activity at 15 minutes and 4 hours. However, intravenous heparin did not attenuate bound Xa/Va activity at 15 minutes or thrombin activity at 15 minutes and 4 hours. Platelet-dependent bound Xa/Va activity occurs rapidly after arterial injury and may promote thrombin elaboration for up to 96 hours. Bound thrombin activity and de novo thrombin elaboration on the vascular wall may play an important role in the progression of thrombosis and vascular wall remodeling.
Increase of Vitamin E Content in LDL and Reduction of Atherosclerosis in Cholesterol-Fed Rabbits by a Water-Soluble Antioxidant-Rich Fraction ofSalvia miltiorrhiza
Authors: Wu Yih-Jer , Hong Chuang-Ye , Lin Shing-Jong , Wu Paulin , Shiao Ming-Shi
Published: June 2011

Abstract—Antioxidants that prevent LDL from oxidation may reduce atherosclerosis.Salvia miltiorrhizaBunge is a Chinese herb widely used for the treatment of atherosclerosis-related disorders. Salvianolic acid B (Sal B), a water-soluble polyphenolic antioxidant isolated from the roots of this plant, was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radicals and inhibit LDL oxidation more effectively than probucol. In order to evaluate the antiatherogenic potential, New Zealand White rabbits were fed for 12 weeks a normal diet, a high cholesterol diet, a high cholesterol diet containing 1% probucol, or a high cholesterol diet containing a 5% water-soluble extract ofS miltiorrhiza(SM). Both SM and probucol feeding reduced plasma cholesterol. LDLs from the SM-treated group were more resistant to Cu2+-induced oxidation and contained more vitamin E (21.7±2.1 nmol/μmol LDL cholesterol) than did LDLs from the high cholesterol diet group (9.6±1.8 nmol/μmol LDL cholesterol) (P<.005). Endothelial damage, determined at week 6, was reduced by 53% in the SM group (P<.01). SM treatment reduced the atherosclerotic area in the abdominal aorta by 56% (P<.005) and cholesterol deposition in the thoracic aorta by 50% (P<.005). The severity of atherosclerosis in the SM group was significantly reduced after adjustment by using cholesterol exposure as an index of the cholesterol-lowering effect. This study concludes that the reduction of atherosclerosis by SM relies not only on its cholesterol-lowering effect but more heavily on its antioxidant potential to prevent endothelial damage and inhibit LDL oxidative modification in hypercholesterolemic animals.
β-Fibrinogen Gene Polymorphism (C148→T) Is Associated With Carotid Atherosclerosis
Authors: Schmidt Helena , Schmidt Reinhold , Niederkorn Kurt , Horner Susanne , Becsagh Peter , Reinhart Birgit , Schumacher Martin , Weinrauch Viktor , Kostner Gert M.
Published: June 2011

Abstract—Polymorphisms at the β-fibrinogen locus have been shown to be associated with plasma concentration of fibrinogen and coronary heart disease. The effect of the genetic heterogeneity of fibrinogen on carotid atherosclerosis has not been determined so far. We examined the influence of the C148→T polymorphism on carotid disease in a large cohort of middle-aged to elderly subjects without evidence of neuropsychiatric disease. This polymorphism is located close to the consensus sequence of the interleukin-6 element and may represent a functional sequence variant. The genotype of 399 randomly selected, neurologically asymptomatic individuals, aged 45 to 75 years, was determined by denaturing gradient gel electrophoresis. Carotid atherosclerosis was assessed by color-coded duplex scanning and was graded on a five-point scale ranging from 0 (=normal) to 5 (=complete luminal obstruction). TheC/C,C/T,andT/Tgenotypes were noted in 226 (56.6%), 148 (37.1%), and 25 (6.3%) individuals, respectively. TheT/Tgenotype group demonstrated higher grades of carotid atherosclerosis than did theC/CandC/Tgenotypes (P=.003). Logistic regression analysis created a model of independent predictors of carotid atherosclerosis that included apolipoprotein B (odds ratio [OR], 1.17/10 mg/dL), age (OR, 2.46/10 years), lifetime tobacco consumption (OR, 1.03/1000 g), presence of the β-fibrinogen promoterT/Tgenotype (OR, 6.17), plasma fibrinogen concentration (OR, 1.05/10 mg/dL), and cardiac disease (OR, 1.80). These data suggest that the β-fibrinogen promoterT/T148 genotype represents a genetic risk factor for carotid atherosclerosis in the middle-aged to elderly.
Cytokines and Cell Adhesion Molecules in Cerebral Ischemia
Authors: Pantoni Leonardo , Sarti Cristina , Inzitari Domenico
Published: June 2011

Abstract—The possibility of reopening an occluded cerebral artery by means of thrombolysis has renewed interest in a number of the several mechanisms that are active during acute cerebral ischemia. Over recent years, it has become apparent that leukocytes play a central role not only during the healing stage of brain infarction but also during the early phases of cerebral ischemia, when it is postulated that these cells produce harmful effects, particularly in the presence of reperfusion. This review is based on the critical analysis of more than 150 publications dealing with the role of leukocytes and some inflammatory mediators (cytokines, chemokines, and adhesion molecules) in cerebral ischemia. Animal studies indicate that leukocyte involvement is promoted by a variety of inflammatory molecules produced immediately after the onset of cerebral ischemia. Considerable experimental evidence suggests that these mediators play a key role in the progression from ischemia to irreversible injury (ie, cellular death and necrosis). However, the precise role of each molecule alone remains to be further elucidated as well as in relation to the complex network existing among different mediators. Progress in our understanding of the inflammatory mechanisms operating in cerebral ischemia has enabled the testing of new compounds with promising results in animals; in contrast, one recent controlled trial of an anti-leukocyte molecule in acute stroke patients showed negative results. This discrepancy may derive in part from our incomplete understanding of the complexity of the inflammatory mechanisms involved in cerebral ischemia. Our analysis suggests that until sufficient knowledge of the underlying disease mechanisms is acquired, more care should be taken when testing new and potentially efficacious drugs in stroke patients.
Role of Macrophage Glycosaminoglycans in the Cellular Catabolism of Oxidized LDL by Macrophages
Authors: Kaplan Marielle , Williams Kevin Jon , Mandel Hanna , Aviram Michael
Published: June 2011

Abstract
—Macrophage binding sites for oxidized LDL (Ox-LDL) include class A scavenger receptors (SR-As), the CD-36 molecule, and an additional but hitherto unidentified binding site. Because cell-surface glycosaminoglycans (GAGs) were previously shown to be involved in the cellular uptake of native LDL and lipoprotein(a), several strategies to assess the participation of heparan sulfate (HS) and chondroitin sulfate (CS) in macrophage catabolism of Ox-LDL were used. First, incubation of J-774 A.1 macrophage-like cells with either heparinase or chondroitinase, or with both enzymes together, reduced the binding, uptake, and degradation of
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I–Ox-LDL by 20% to 45%, in comparison with control nontreated cells, while catabolism of
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I-labeled acetylated LDL (Ac-LDL) and native LDL were unaffected. Second, the proteoglycan (PG) cellular content was increased by cell enrichment with exogenous GAGs or by using human monocyte-derived macrophages from two patients with Sanfilippo mucopolysaccharidosis, which are characterized by cellular HS accumulation. In these macrophages, cellular uptake of
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I–Ox-LDL increased, while catabolism of
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I–Ac-LDL and native LDL were unaffected. Experiments using conditioned media from control, heparinase-digested, or chondroitinase-digested macrophages indicated that neither secreted GAGs nor released digestion products played any role in Ox-LDL catabolism. To evaluate potential interactions between cell-surface GAGs and known receptors for Ox-LDL, we used excess unlabeled Ac-LDL to block SR-As or anti–CD-36 antibodies to block CD-36, and then examined the catabolism of
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I–Ox-LDL by GAG-enriched or -depleted macrophages. Both excess unlabeled Ac-LDL and anti–CD-36 antibodies reduced
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I–Ox-LDL catabolism, but only excess unlabeled Ac-LDL completely abolished the increase in
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I–Ox-LDL catabolism on GAG enrichment of the cells, indicating a cooperation between exogenous GAGs and cell-surface SR-As in the catabolism of OX-LDL. Moreover, the addition of GAGases to macrophages that were preincubated with anti–CD-36 antibodies and excess Ac-LDL further reduced macrophage degradation of Ox-LDL in comparison with cells that were pretreated only with anti–CD-36 antibodies and Ac-LDL, indicating a more complex role for endogenous GAGs. Overall, these studies demonstrate a substantial contribution of macrophage-associated GAGs in the catabolism of Ox-LDL, which is mediated in part by a cooperation between GAGs and cell-surface SR-As.