bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Superoxide Anions on Endothelial Ca
2+
Signaling Pathways
Authors: Graier Wolfgang F. , Hoebel Bernhard G. , Paltauf-Doburzynska Jolanta , Kostner Gert M.
Published: June 2011

Abstract
—Although the involvement of free radicals in the development of endothelial dysfunction under pathological conditions, like diabetes and hypercholesterolemia, has been proposed frequently, there is limited knowledge as to how superoxide anions (O
2
−
) might affect endothelial signal transduction. In this study, we investigated the effects of preincubation with the O
2
−
-generating system xanthine oxidase/hypoxanthine (XO/HX) on mechanisms for Ca
2+
signaling in cultured porcine aortic endothelial cells. Incubation of cells with XO/HX yielded increased intracellular Ca
2+
release and capacitative Ca
2+
entry in response to bradykinin and ATP in a time- and concentration-dependent manner. This effect was prevented by superoxide dismutase but not by the tyrosine kinase inhibitor tyrphostin A48. In addition, capacitative Ca
2+
entry induced by the receptor-independent stimulus 2,5-di-(
tert
-butyl)-1,4-benzohydroquinone or thapsigargin was enhanced in O
2
−
-exposed cells (+38% and +32%, respectively). Increased Ca
2+
release in response to bradykinin in XO/HX-pretreated cells might be due to enhanced formation of inositol-1,4,5-trisphosphate (+140%). Exposure to XO/HX also affected other signal transduction mechanisms involved in endothelial Ca
2+
signaling, such as microsomal cytochrome P450 epoxygenase and membrane hyperpolarization to Ca
2+
store depletion with thapsigargin (+103% and +48%, respectively) and tyrosine kinase activity (+97%). A comparison of bradykinin-initiated intracellular Ca
2+
release and thapsigargin-induced hyperpolarization with membrane viscosity modulated by XO/HX (decrease in viscosity) or cholesterol (increase in viscosity) reflected a negative correlation between bradykinin-initiated Ca
2+
release and membrane viscosity. Because intracellular Ca
2+
is a main regulator of endothelial vascular function, our data suggest that O
2
−
anions are involved in regulation of the vascular endothelium.
Dietary Antioxidants Inhibit Development of Fatty Streak Lesions in the LDL Receptor–Deficient Mouse
Authors: Crawford Richard S. , Kirk Elizabeth A. , Rosenfeld Michael E. , LeBoeuf Renée C. , Chait Alan
Published: June 2011

Abstract—Oxidized low density lipoprotein (LDL) promotes atherogenesis. Although pharmacological antioxidants such as probucol inhibit both LDL oxidation and atherosclerosis in hyperlipidemic animals, the effects of natural antioxidants such as vitamin E are inconclusive. To further determine the effects of supplemental dietary antioxidants in vivo, we evaluated whether combined dietary antioxidants (0.1% vitamin E, 0.5% β-carotene, and 0.05% vitamin C) inhibit LDL oxidation and fatty streak lesion development in homozygous LDL receptor–null (LDLR−/−) mice fed a high-fat, high-cholesterol diet. An additional group of mice were fed black tea, which has been shown to inhibit LDL oxidation in vitro. After receiving a high-fat, high-cholesterol diet for 8 weeks, the combined antioxidant–supplemented (antioxidant) group (n=18), tea group (n=19), and control group (n=17) had equivalent plasma cholesterol levels. LDL oxidation, as measured by the lag phase of conjugated diene formation, was markedly inhibited in the antioxidant group compared with the tea or control groups [mean lag phases=143±7 (antioxidant), 100±5 (tea), and 84±4 (control) minutes;P<0.0001 antioxidant versus tea or control]. The cross-sectional surface area of fatty streak lesions in the aortic sinus was reduced by 60% in the antioxidant group compared with both the tea and control groups (P<0.0001 antioxidant versus tea or control). There was no difference in lesion area between tea and control groups. Although both LDL oxidation and atherosclerosis were significantly inhibited in the antioxidant group, no correlation between lag phase values and lesion size was observed among individual animals. Furthermore, black tea did not inhibit fatty streak development in LDLR−/−mice. These data suggest that combined natural dietary antioxidants inhibit both LDL oxidation and atherogenesis in animals with elevated LDL but that inhibition of LDL oxidation alone may not prevent the development of atherosclerosis.
Apoptosis
Authors: Best Patricia J. M. , Hasdai David , Sangiorgi Giuseppe , Schwartz Robert S. , Holmes David R. , Simari Robert D. , Lerman Amir
Published: June 2011

Abstract
—Apoptosis is an active form of cell death that is intricately regulated and distinct from necrosis. Data suggest that apoptosis may play a role in the pathophysiology of coronary atherosclerotic disease. Anatomic evidence of apoptosis has been observed in coronary atherosclerosis, restenosis, and transplant arteriopathy, accompanied by an increase in biochemical and genetic markers of apoptosis. Vasoactive substances such as nitric oxide and angiotensin II also regulate vascular smooth muscle cell apoptosis; vasodilating factors may induce apoptosis, whereas vasoconstricting factors may inhibit apoptosis. The aim of this article is to review key points regarding the detection of apoptosis, its regulation, and its possible role in the pathogenesis of coronary artery disease.
Autoantibodies Against Oxidized Low Density Lipoprotein in Patients With Angiographically Verified Coronary Artery Disease
Authors: Lehtimäki Terho , Lehtinen Saara , Solakivi Tiina , Nikkilä Matti , Jaakkola Olli , Jokela Hannu , Ylä-Herttuala Seppo , Luoma Jukka S. , Koivula Timo , Nikkari Tapio
Published: June 2011

Abstract
—Oxidation of low density lipoproteins (LDL) obviously plays an important role in the pathogenesis of atherosclerosis. The purpose of the study was to determine whether antibodies against oxidized LDL are associated with coronary artery disease (CAD). We determined the serum levels of antibodies against copper-oxidized LDL by enzyme-linked immunosorbent assay in 58 patients with angiographically verified CAD and 34 controls without CAD. The mean antibody level, expressed in optical density units, was significantly higher in patients than in controls (0.150±0.088 versus 0.094±0.054, respectively;
P
=0.00089). In logistic regression analysis, high antibody level against oxidized LDL was associated significantly with CAD (
P
=0.0114), independent of age (
P
=0.00137), gender (
P
=0.0021), body mass index (
P
=0.5947), triglyceride concentration (
P
=0.9813), and total cholesterol–high density lipoprotein (HDL) cholesterol (
P
=0.0080) group. Similar analysis in nondiabetic subjects (n=79) and in men only (n=75) showed analogous results, with only minor changes in
P
values. The antibody level against oxidized LDL differed significantly between nonsmokers and smokers in CAD patients (
P
<0.00197) but not in controls (
P
=NS). In addition, the antibody level against oxidized LDL differed significantly between nonsmokers and smokers in subjects with low HDL cholesterol (≤0.9 mmol/L) but not in subjects with high HDL cholesterol (>0.9 mmol/L). In conclusion, elevated levels of antibodies against oxidized LDL were associated with CAD. The data suggest that oxidized LDL plays a role in the pathogenesis of atherosclerosis and suggest a protective function for HDL against LDL oxidation.
Growth and Cell Cycle Abnormalities of Fibroblasts From Tangier Disease Patients
Authors: Drobnik Wolfgang , Liebisch Gerhard , Biederer Carola , Trümbach Barbara , Rogler Gerhard , Müller Peter , Schmitz Gerd
Published: June 2011

Abstract—We have investigated the abnormal proliferation and morphology of fibroblasts from patients with Tangier disease (TD), a high density lipoprotein (HDL) deficiency syndrome that is characterized by impairment of HDL3-mediated lipid efflux and Gi-protein–mediated signaling via phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase D (PLD). TD fibroblasts displayed a 30% to 50% reduced in vitro growth rate and a 1.6-fold increased cell surface area. The response to different mitogens was diminished, and asynchronously growing TD fibroblasts showed 4.4±0.3% S-phase and 19.1±0.5% G2/M-phase cells compared with 9.7±0.6% and 7.8±0.5%, respectively, in controls. Monensin, but not brefeldin A, induced an S- and G2/M-phase distribution in control cells similar to that found in TD fibroblasts. This effect of monensin was accompanied by an increase of ceramide levels in controls, whereas TD fibroblasts already had a 2.5-fold increased basal ceramide concentration. Incubation of control cells with C2 ceramide andthreo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) mimicked the effect of monensin on the cell cycle. The inhibition of neither Giprotein function by pertussis toxin nor PLD by butanol resulted in a G2/M-phase arrest. Propranolol, known to increase phosphatidic acid levels, was ineffective in reversing the G2/M-phase arrest in TD fibroblasts. In addition, cDNA sequences and mRNA expression of the participants of PI-PLC or PLD signaling, ie, G-protein subunits αi1, αi2, and αi3; phosphatidylinositol transfer proteins-α and -β; and ADP ribosylation factors 1 and 3 were found to be normal. Thus, growth and cell cycle abnormalities in TD fibroblasts are likely to be related to impaired Golgi function and sphingolipid signaling rather than inoperative G-protein signal transduction. Because PDMP was also found to decrease HDL3-mediated lipid efflux in control but not TD fibroblasts, similar pathways seem to be involved in the disturbances of lipid transport and growth retardation.
On the Role of c-Jun in the Induction of PAI-1 Gene Expression by Phorbol Ester, Serum, and IL-1α in HepG2 Cells
Authors: Arts Janine , Grimbergen Jos , Toet Karin , Kooistra Teake
Published: June 2011

Abstract
—We have characterized the regulation of plasminogen activator inhibitor-1 (PAI-1) gene expression by phorbol 12-myristate 13-acetate (PMA), serum, and interleukin-1α (IL-1α) in the human hepatoma cell line HepG2. PMA, serum, and IL-1α induced a rapid and transient 28-fold (PMA), 9-fold (serum), and 23-fold (IL-1α) increase in PAI-1 mRNA, peaking after ≈4 hours. These inductions of PAI-1 mRNA accumulation were reduced by pretreatment of the HepG2 cells with the protein tyrosine kinase inhibitor genistein. Conversely, stimulation of tyrosine phosphorylation by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in PAI-1 mRNA levels. The effects of PMA, serum, and IL-1α on PAI-1 mRNA expression have been compared with their ability to modulate the expression of a chloramphenicol acetyltransferase (CAT) reporter plasmid, which was under control of the −489 to +75 region of the PAI-1 promoter, and stably transfected into HepG2 cells. This region of the PAI-1 promoter was previously found to contain a tetradecanoyl phorbol acetate–response element (TRE; between −58 and −50) necessary for PMA responsiveness and with a high affinity for c-Jun homodimers. Whereas incubation of these transfected HepG2 cells with PMA and serum showed an induction profile of CAT mRNA similar to that of PAI-1 mRNA, hardly any induction of CAT mRNA was found with IL-1α. In line with these findings, IL-1α poorly induced c-Jun homodimer binding to the PAI-1 TRE in gel mobility-shift assays. Pretreatment of HepG2 cells with the protein kinase C inhibitor Ro 31-8220 or the mitogen-activated protein kinase kinase (MAPKK)
1,2
activity blocker PD98059 selectively suppressed the induction of PAI-1 (and CAT) expression by PMA, but not that by IL-1α. In contrast, the protein tyrosine kinase inhibitor herbimycin A blocked PAI-1 mRNA induction by IL-1 α only. We propose 2 separate PAI-1 inductory pathways for PMA and IL-1α in HepG2, both involving protein tyrosine kinase activation; the serum-induced signaling pathway may (partially) overlap with the PMA-activated protein kinase C/mitogen-activated protein kinase kinase pathway, leading to c-Jun homodimer binding to the PAI-1 TRE.
Tissue Factor Expression of Human Monocytes Is Suppressed by Lysophosphatidylcholine
Authors: Engelmann Bernd , Zieseniss Susanne , Brand Korbinian , Page Sharon , Lentschat Arnd , Ulmer Artur J. , Gerlach Eckehart
Published: June 2011

Abstract
—The expression of tissue factor (TF), the principal initiator of coagulation, is increased during inflammation and atherosclerosis. Both conditions are promoted by lysophosphatidylcholine (lysoPC). We observed in the present study that lysoPC (1 to 10 μmol/L) dose-dependently reduced TF activity in human monocytes, as elicited by lipopolysaccharide (LPS). Lysophosphatidylethanolamine (lysoPE) and other lysophospholipids did not affect LPS-induced TF activity of human monocytes. TF antigen expression as elicited by LPS was also lowered by lysoPC. Phospholipid analyses indicated a selective increase in the lysoPC content of the monocytes after preincubation with the lysophospholipid. LysoPC inhibited the TF activity of Mono Mac-6 cells to a similar extent as in the monocytes. LPS binding to plasma membrane receptors and internalization of LPS into monocytes were not affected by lysoPC. In contrast, LPS-mediated nuclear binding of nuclear factor-κB/Rel to a TF-specific κB site was inhibited by lysoPC. Induction of TF mRNA expression by LPS tended to be partially reduced by the lysophospholipid. Preincubation with lysoPC increased monocytic cAMP levels. Inhibition of adenylyl cyclase by pretreatment with 2′-deoxy-3′-adenosine monophosphate partially reversed the inhibition of TF activity promoted by lysoPC. In conclusion, lysoPC markedly decreases LPS-mediated TF expression of human monocytes, the effect probably being mediated by both transcriptional and posttranscriptional mechanisms. LysoPC may thus attenuate activation of coagulation during inflammation and atherosclerosis.
Dietary ω-3, ω-6, and ω-9 Unsaturated Fatty Acids and Growth Factor and Cytokine Gene Expression in Unstimulated and Stimulated Monocytes
Authors: Baumann Klaus H. , Hessel Franz , Larass Iris , Müller Thomas , Angerer Peter , Kiefl Rosemarie , von Schacky Clemens
Published: June 2011

Abstract—Dietary ω-3 fatty acids retard coronary atherosclerosis. Previously, we demonstrated that dietary ω-3 fatty acids reduce platelet-derived growth factor (PDGF)-A and PDGF-B mRNA levels in unstimulated, human mononuclear cells (MNCs). In a randomized, investigator-blinded intervention trial, we have now compared the effect of ingestion of 7 g/d ω-3, ω-6, or ω-9 fatty acids for 4 weeks versus no dietary intervention on PDGF-A, PDGF-B, heparin-bound epidermal growth factor (HB-EGF), monocyte chemoattractant protein-1 (MCP-1), and interleukin-10 gene expression in unstimulated MNCs and in monocytes that were adherence-activated ex vivo in a total of 28 volunteers. In unstimulated MNCs, mRNA steady-state levels of PDGF-A, PDGF-B, and MCP-1 were reduced by 25±10%, 31±13%, and 40±14%, respectively, after ω-3 fatty acid ingestion, as assessed by quantitative polymerase chain reaction (allP<0.05). In monocytes that were adherence-activated ex vivo for 4 and 20 hours, mRNA steady-state levels of PDGF-A, PDGF-B, and MCP-1 were reduced by 25±13%, 20±15%, and 30±8%, respectively (allP<0.05). Interleukin-10 and HB-EGF mRNA steady-state levels were not influenced by ω-3 fatty acid ingestion. Expression of all respective mRNAs in control volunteers or in those ingesting ω-6 or ω-9 fatty acids were not altered. We conclude that human gene expression for PDGF-A, PDGF-B, and MCP-1, factors thought relevant to atherosclerosis, is constitutive, is constant, and can be reduced only by dietary ω-3 fatty acids in unstimulated and adherence-activated monocytes.
Relationship Between Plasma Fibrinogen and Coronary Heart Disease in Women
Authors: Eriksson Margita , Egberg Nils , Wamala Sarah , Orth-Gomér Kristina , Mittleman Murray A. , Schenck-Gustafsson Karin
Published: June 2011

Abstract
—Plasma fibrinogen is an independent risk factor for coronary heart disease (CHD) in men; however, its role in women is less clear. We examined the ability of plasma fibrinogen to predict CHD in a community-based, case-control study of women aged 65 years or younger living in the greater Stockholm area. Cases were all patients hospitalized for an acute coronary event between February 1991 and February 1994. Controls were randomly selected from the city census and were matched to cases by age and catchment area. Plasma fibrinogen was measured 3 to 6 months after hospitalization by using a fibrinogen assay based on fibrinogen polymerization time measurement. Of the 292 consecutive cases, 110 (37%) were hospitalized for an acute myocardial infarction and 182 (63%) for angina pectoris. The mean age±SD in both patients and controls was 56±7 years. Mean levels of plasma fibrinogen in patients and controls were 3.66±0.81 and 3.25±0.64 g/L (
P
<0.0001), respectively. The age-adjusted odds ratio (OR) for CHD in the highest versus the lowest quartile of plasma fibrinogen was 6.0 (95% confidence interval [CI], 3.5 to 10.4). After adjustment for age, cigarette smoking, body mass index, systolic blood pressure, total cholesterol, high density lipoprotein cholesterol, triglycerides, and educational level, the OR was 3.0 (95% CI, 1.6 to 5.5). Further adjustment for C-reactive protein yielded the same result. In both premenopausal and postmenopausal women, the multivariate-adjusted ORs were 7.0 (95% CI, 1.8 to 28.3) and 2.1 (95% CI, 1.0 to 4.4), respectively. These results provide evidence that plasma fibrinogen is associated with an excess risk of CHD in women.
Central Role of the MAPK Pathway in Ang II–Mediated DNA Synthesis and Migration in Rat Vascular Smooth Muscle Cells
Authors: Xi Xiao-Ping , Graf Kristof , Goetze Stephan , Fleck Eckart , Hsueh Willa A. , Law Ronald E.
Published: June 2011

Abstract
—Angiotensin II (Ang II) promotes vascular smooth muscle cell (VSMC) growth and migration, but the signaling pathways mediating these VSMC behaviors critical to restenosis and atherosclerosis are not completely known. The purpose of the present investigation was to define the role of mitogen-activated protein kinase (MAPK) in Ang II–induced DNA synthesis, migration, and c-
fos
induction in VSMCs. PD 98059, a synthetic inhibitor of MAPK kinase, or antisense oligodeoxynucleotides (ODNs) to deplete extracellular signal–regulated kinase (ERK)1 and ERK2 MAPKs, were used to inhibit MAPK signaling. PD 98059 at 30 μmol/L reduced Ang II–induced MAPK activity by 69% (
P
<0.01). Under these conditions, Ang II–induced DNA synthesis was completely inhibited (
P
<0.01), and Ang II–directed migration was attenuated by 76% (
P
<0.05). In contrast, induction of c
-fos
by Ang II was only partially suppressed (58% inhibition,
P
<0.01). Antisense ODNs against the initiation site of rat ERK1 and ERK2 MAPK mRNAs reduced corresponding protein levels by 63% (
P
<0.01) and completely inhibited MAPK activation by either Ang II (1 μmol/L) or 10% serum. Antisense ODNs (0.4 μmol/L) completely inhibited Ang II–induced DNA synthesis (
P
<0.01), decreased migration by 47% (
P
<0.01), and reduced c-
fos
induction by 40% (
P
<0.01 versus control ODN–transfected VSMCs). The Ang II type 1 (AT
1
)-receptor blocker irbesartan completely blocked DNA synthesis, migration, MAPK activation, and c-
fos
induction by Ang II in VSMCs. These results demonstrate that activation of MAPK plays a crucial role in Ang II–directed migration and DNA synthesis through the AT
1
receptor. In contrast, Ang II–mediated c-
fos
induction and migration were only partially inhibited by either antisense ODNs or PD 98059, suggesting that other pathways in addition to the MAPK pathway may be involved in these actions of Ang II. We conclude that MAPK is a critical regulatory factor for Ang II–mediated migration and growth in VSMCs. Ang II–induced DNA synthesis showed a stronger MAPK dependence than did Ang II–directed migration or c-
fos
induction.
ADP Receptors and Clinical Bleeding Disorders
Authors: Cattaneo Marco , Gachet Christian
Published: June 2011

Abstract—ADP plays a key role in hemostasis and thrombosis. Despite its early identification in 1961 as the first known aggregating agent, the molecular basis of ADP-induced platelet activation is only beginning to be understood. The present review proposes a model of 3 purinergic receptors contributing separately to the complex process of ADP-induced platelet aggregation: the P2X1ionotropic receptor, responsible for rapid influx of ionized calcium into the cytosol; the P2Y1metabotropic receptor, responsible for mobilization of ionized calcium from internal stores, which initiates aggregation; and an as-yet-unidentified P2Y receptor coupled to Gαi2, which is essential for the full aggregation response to ADP. It is probable that this as-yet-unidentified receptor is the molecular target of the ADP-selective antiaggregating drugs ticlopidine and clopidogrel. In addition, it is probably defective in patients with a bleeding diathesis that is characterized by selective impairment of platelet responses to ADP.
Histamine Induces Tyrosine Phosphorylation of Endothelial Cell-to-Cell Adherens Junctions
Authors: Andriopoulou Paraskevi , Navarro Pilar , Zanetti Adriana , Lampugnani Maria Grazia , Dejana Elisabetta
Published: June 2011

Abstract
—Endothelial adherens junctions (AJ) promote intercellular adhesion and may contribute to the control of vascular permeability. These structures are formed by a transmembrane and cell-specific adhesive protein, vascular endothelial (VE)-cadherin, which is linked by its cytoplasmic tail to intracellular proteins called catenins (α-catenin, β-catenin, and plakoglobin) and to the actin cytoskeleton. Little is known about the functional regulation of AJ in endothelial cells. In this study, we analyzed the effect of histamine on AJ organization in cultured endothelial cells. We first observed that histamine induced detectable intercellular gaps only in loosely-confluent cells, whereas this effect was strongly reduced or absent in long-confluent cultures. Despite this difference, in vitro permeability was augmented by histamine in both conditions. In resting conditions, tyrosine phosphorylation of AJ components and permeability values were higher in recently-confluent cells as compared with long-confluent cells. Histamine did not affect the phosphorylation state of AJ in recently-confluent cells but strongly increased this parameter in long-confluent cultures. In addition, in long-confluent cells, histamine caused dissociation of VE-cadherin from the actin cytoskeleton measured by a decrease of the amount of the molecule in the detergent-insoluble fraction of the cell extracts. Dibutyryl cAMP was able to prevent the effect of histamine on both tyrosine phosphorylation of AJ components and on endothelial permeability. The effect of histamine was specific for VE-cadherin because the phosphorylation state of neural (N)-cadherin, the other major endothelial cadherin, was unchanged by this agent. Hence AJ components are a target of histamine activation cascade; we suggest that induction of tyrosine phosphorylation of VE-cadherin and catenins contributes to the histamine effect on permeability, even in absence of frank intercellular gaps and cell retraction.
Expression of the Angiogenic Protein, Platelet-Derived Endothelial Cell Growth Factor, in Coronary Atherosclerotic Plaques
Authors: Ignatescu Mihaela C. , Gharehbaghi-Schnell Elisabeth , Hassan Ali , Rezaie-Majd Shahrzad , Korschineck Irina , Schleef Raymond R. , Glogar Helmut D. , Lang Irene M.
Published: June 2011

Abstract
—Recent information indicates that platelet-derived endothelial cell growth factor (PD-ECGF), a 45-kDa angiogenic protein, is expressed in the endothelium of various tissues and that its level of expression is correlated with the number of microvessels in human tumors. Because the formation of neovessels is also thought to play a role in atherosclerotic vascular remodeling, we analyzed PD-ECGF expression in fresh, coronary plaque tissues obtained by directional coronary atherectomy. Specimens from 31 patients were collected and analyzed by reverse transcription–polymerase chain reaction, histochemical staining, immunohistochemistry, and in situ hybridization with the use of PD-ECGF–specific primers and probes. Lesional vascular remodeling was assessed by intravascular ultrasound. PD-ECGF immunoreactivity and mRNA were found in plaque macrophages, endothelial cells of plaque neovessels, and stellate smooth muscle cells of 20 atherectomy specimens (64.5%). PD-ECGF immunoreactivity was correlated with the number of lesional microvessels and mast cells. Double-staining experiments revealed a close spatial proximity of PD-ECGF–positive cells and mast cells. Furthermore, the numbers of microvessels and mast cells were significantly higher in lesions lacking compensatory enlargement. The data indicate that PD-ECGF is expressed within cells of the atherosclerotic plaque and may be involved in driving angiogenesis in concert with mast cells.
Systemic Inflammatory Parameters in Patients With Atherosclerosis of the Coronary and Peripheral Arteries
Authors: Erren Michael , Reinecke Holger , Junker Ralf , Fobker Manfred , Schulte Helmut , Schurek Josef O. , Kropf Jürgen , Kerber Sebastian , Breithardt Günter , Assmann Gerd , Cullen Paul
Published: June 2011

Abstract
—Plasma concentration of markers of inflammation are increased in patients with atherosclerosis. However, it is unclear whether the pattern and magnitude of this increase vary with the site and extent of disease. In 147 patients undergoing semiquantitative coronary angiography, we measured the acute-phase reactants C-reactive protein (CRP) or serum amyloid A (SAA); the proinflammatory cytokine interleukin 6 (IL-6); the active and total fractions of the anti-inflammatory cytokine transforming growth factor-β (TGF-β); the macrophage activation marker neopterin; and the infection marker procalcitonin. Compared with 62 patients without either coronary artery disease (CAD) or peripheral artery disease (PAD), 57 patients with CAD but no PAD showed greater median CRP (0.4 versus 0.2 mg/dL,
P
=0.004) and IL-6 (3.8 versus 1.6 pg/mL,
P
=0.007) levels and a lower level of active-TGF-β (57 versus 100 ng/mL,
P
=0.038). Moreover, CRP, IL-6, and neopterin levels showed a positive and the active TGF-β level a negative correlation with the extent of coronary atherosclerosis. Compared with these 57 patients with CAD alone, 15 patients with PAD and CAD had higher median levels of SAA (17 versus 7 mg/mL,
P
=0.008), IL-6 (12 versus 4 pg/mL,
P
=0.002), neopterin (14 versus 11 mg/dL,
P
=0.006), and total TGF-β (11834 versus 6417 ng/L,
P
=0.001). However, these strong univariate associations of markers of inflammation and atherosclerosis were lost in multivariate analysis once age, sex, and high density lipoprotein cholesterol or fibrinogen were taken into account. Increased plasma levels of CRP, SAA, IL-6, TGF-β, neopterin, and procalcitonin constitute an inflammatory signature of advanced atherosclerosis and are correlated with the extent of disease but do not provide discriminatory diagnostic power over and above established risk factors.
Detection, Quantification, and Characterization of Potentially Atherogenic Triglyceride-Rich Remnant Lipoproteins
Authors: Cohn Jeffrey S. , Marcoux Caroline , Davignon Jean
Published: June 2011

Abstract
—Triglyceride-rich lipoprotein (TRL) remnants are formed in the circulation when apolipoprotein (apo) B-48–containing chylomicrons of intestinal origin or apoB-100–containing VLDL of hepatic origin are converted by lipoprotein lipase, and to a lesser extent by hepatic lipase, into smaller and more dense particles. Compared with their nascent precursors, TRL remnants are depleted of triglyceride, phospholipid, and C apolipoproteins and are enriched in cholesteryl esters and apoE. They can thus be identified, separated, and/or quantified in plasma according to their density, charge, size, specific lipid components, apolipoprotein composition, and/or apolipoprotein immunospecificity. Each of these approaches has contributed to our current understanding of the compositional characteristics of TRL remnants and their potential to promote atherosclerosis. An ongoing search is nevertheless under way for more accurate and clinically applicable remnant lipoprotein assays that will be able to better define coronary artery disease risk in patients with hypertriglyceridemia.
Vitamin E Inhibits Collagen-Induced Platelet Activation by Blunting Hydrogen Peroxide
Authors: Pignatelli Pasquale , Pulcinelli Fabio M. , Lenti Luisa , Gazzaniga Pier Paolo , Violi Francesco
Published: June 2011

Abstract
—In this study, we investigated whether vitamin E at concentrations achievable in blood after supplementation inhibits platelet function in humans. Gel-filtered platelets were incubated 30 minutes with scalar concentrations (50 to 250 mmol/L) of vitamin E and then stimulated with collagen. Compared with controls, vitamin E inhibited collagen-induced platelet aggregation and thromboxane A2 formation in a dose-dependent manner. Furthermore, vitamin E inhibited, in a dose-dependent manner, Ca
2+
mobilization and formation of inositol 1,4,5-triphosphate. Because it was previously shown that hydrogen peroxide formation mediates arachidonic acid metabolism and phospholipase C activation in collagen-induced platelet activation, we investigated whether vitamin E was able to blunt hydrogen peroxide. In experiments performed in unstimulated platelets supplemented with hydrogen peroxide and in collagen-stimulated platelets, vitamin E was able to blunt hydrogen peroxide. In 6 healthy subjects given vitamin E for 2 weeks (600 mg/d), we found a significant decrease of collagen-induced H
2
O
2
formation, platelet aggregation, and calcium mobilization. This study demonstrated in vitro and ex vivo that vitamin E inhibits collagen-induced platelet activation by blunting hydrogen peroxide formation.
LDL Size and Risk of Coronary Heart Disease in Elderly Men and Women
Authors: Mykkänen Leena , Kuusisto Johanna , Haffner Steven M. , Laakso Markku , Austin Melissa A.
Published: June 2011

Abstract
—A predominance of small, dense, low density lipoprotein (LDL) particles has consistently been associated with coronary heart disease (CHD) in young and middle-aged subjects in cross-sectional studies. Recently, 3 prospective, case-control studies showed that decreased LDL size is a predictor of CHD in middle-aged subjects. However, it is not known whether decreased LDL size is mainly associated with premature CHD or whether it continues to play a role in CHD risk at older ages also. We performed a prospective, nested case-control study in 86 subjects (58 nondiabetic and 28 type 2 diabetic) aged 65 to 74 years who were free of myocardial infarction at baseline and who then had a myocardial infarction or CHD death during a 3.5-year follow-up (cases) and in 172 controls matched for sex and diabetes status but who remained free of CHD during follow-up. LDL particle size determined by gradient gel electrophoresis (268.2±0.9 versus 268.5±0.7 Å,
P
=0.782) and the proportion of subjects with LDL subclass phenotype B (20.9 versus 21.5,
P
=0.914) were similar among cases and controls. Furthermore, diastolic blood pressure, total cholesterol, high density lipoprotein cholesterol, triglycerides, apolipoprotein A
1
, fasting glucose, fasting insulin, waist-to-hip ratio, and body mass index were not associated with CHD risk. However, smoking and increased systolic blood pressure, apolipoprotein B levels, and the total cholesterol–high density lipoprotein cholesterol ratio were significant predictors of CHD events both in univariate and multivariate analyses. Our findings indicate that LDL size is not a predictor of CHD events in elderly white subjects after controlling for diabetes status.
Hypertension and Endothelial Dysfunction in Apolipoprotein E Knockout Mice
Authors: Yang Renhui , Powell-Braxton Lyn , Ogaoawara Annie Ko , Dybdal Noel , Bunting Stuart , Ohneda Osamu , Jin Hongkui
Published: June 2011

Abstract
—Mice lacking ApoE (
Apoe
–/–
) develop initially hypercholesterolemia and latterly atherosclerosis. This study examined hemodynamics and endothelial function in 6-week-old
Apoe
–/–
mice with hypercholesterolemia only, 7.5-months-old
Apoe
–/–
mice with both hypercholesterolemia and atherosclerosis, and age matched controls. One day after implantation of catheters into the carotid artery, arterial pressure was measured in conscious, unrestrained mice. Compared with the respective controls, there was a significant increase in arterial pressure and the ratio of left ventricular weight to body weight in 7.5-month-old
Apoe
–/–
mice but not in 6-week-old
Apoe
–/–
mice. Histopathological analysis demonstrated significant renal artery disease in the form of extensive atheromatous plaques only in 7.5-month-old
Apoe
–/–
mice, whereas no atherosclerotic lesions were found in 6-week-old
Apoe
–/–
mice. For evaluation of endothelial function, a laser Doppler perfusion imager with a computer-controlled optical scanner was used to measure cutaneous blood perfusion on the dorsal side of one hind paw before and after topical application of mustard oil, which is known to induce nitric oxide–mediated vasodilation. The mustard oil treatment elicited a substantial increase in blood perfusion (
P
<0.01), which was similar between 6-week-old
Apoe
–/–
mice and controls but significantly blunted in 7.5-month-old
Apoe
–/–
mice versus control mice, suggesting nitric oxide–mediated vasodilation is diminished in 7.5-month-old
Apoe
–/–
mice but not in 6-week-old
Apoe
–/–
mice. In contrast, the increase in blood perfusion induced by topical administration of cilostazol, which induces vasodilation via cyclic adenosine monophosphate, was not different between 7.5-month-old
Apoe
–/–
mice and controls. Thus hypertension and endothelial dysfunction observed in 7.5-month-old
Apoe
–/–
mice may be due mainly to atherosclerosis.
Effects of Hypercholesterolemia on Myocardial Ischemia-Reperfusion Injury in LDL Receptor–Deficient Mice
Authors: Girod Wesley G. , Jones Steven P. , Sieber Nola , Aw Tak Yee , Lefer David J.
Published: June 2011

Abstract
—Hypercholesterolemia is a primary risk factor for atherosclerosis, coronary artery disease, and myocardial infarction. We subjected low density lipoprotein receptor–deficient (LDLr –/–) and control (wild-type) mice to 30 minutes of myocardial ischemia and 120 minutes of reperfusion. Myocardial infarction per area at risk (AAR) was noted under baseline conditions to be significantly (
P
<0.05) smaller in the LDLr –/– mice compared with wild-type mice (24.7±3.2% and 38.8±4.3% of AAR, respectively). Subsequently, mice were fed a high-cholesterol diet (HCD) for 2 or 12 weeks, which resulted in significant increases in serum cholesterol levels in both LDLr –/– and wild-type groups. After 2 weeks of the HCD, the LDLr –/– mice demonstrated a significant elevation (
P
<0.01) in myocardial necrosis per AAR (50.2±5.36% of AAR) compared with the normal-diet LDLr –/– group, whereas the short-term HCD-fed wild-type mice demonstrated no significant difference from baseline. In contrast, wild-type mice fed the HCD for 12 weeks revealed a significant (
P
<0.05) decrease in necrosis per AAR, which was 22.5±3.2% of the AAR in comparison with that in the normal-diet wild-type mice (38.8±4.3% of AAR). LDLr –/– mice on the same long-term HCD showed a similar significantly (
P
<0.05) decreased infarct size, which was 13.2±4.0% of the AAR. In additional experiments, we determined that myocardial tissue total glutathione (GSH) levels were reduced after 2 weeks of the HCD and were significantly increased after 12 weeks of the HCD in the LDLr –/– mouse heart. These data suggest that short-term cholesterol feeding renders the myocardium of LDLr –/– mice more susceptible to ischemia-reperfusion injury, whereas more long-term hypercholesterolemia confers cardioprotection in the LDLr –/– mouse heart.
Interleukin-10 Blocks Atherosclerotic Events In Vitro and In Vivo
Authors: Pinderski Oslund Laura J. , Hedrick Catherine C. , Olvera Tristana , Hagenbaugh Amy , Territo Mary , Berliner Judith A. , Fyfe Alistair I.
Published: June 2011

Abstract—Atherosclerosis can be viewed in part as an inflammatory disease process and may therefore be susceptible to manipulation of the immune state. Interleukin 10 (IL-10) is an inhibitory cytokine produced by activated lymphocytes and monocytes. These studies present evidence that IL-10 can inhibit minimally oxidized LDL (MM-LDL)–induced monocyte-endothelium interaction as well as inhibit atherosclerotic lesion formation in mice fed an atherosclerotic diet. Pretreatment of human aortic endothelial cells (HAECs) for 18, but not 4, hours with recombinant IL-10 caused a significant decrease in MM-LDL–induced monocyte binding. IL-10 was found to be maximally effective at 10 ng/mL. Transfection of HAECs with adenovirus expressing viral bcrf-1 IL-10 (Ad-vIL-10) in a sense but not antisense orientation completely inhibited the ability of MM-LDL to induce monocyte binding. Similar results were obtained with IL-10 or Ad-vIL-10 in HAECs stimulated with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). We have previously shown increases in cAMP associated with MM-LDL activation of endothelial cells. The MM-LDL–induced increase in cAMP levels was not inhibited by preincubation with IL-10. In vivo studies demonstrated that mice with a murine IL-10 transgene under the control of the human IL-2 promoter have decreased lesions versus controls on an atherogenic diet (5433±4008 mm2versus 13 574±4212 mm2;P<0.05), whereas IL-10 null mice have increased lesions (33 250±9117 mm2;P<0.0001) compared with either controls or IL-10 transgenic mice. These studies suggest an important role for IL-10 in the atherosclerotic disease process.
Augmented Expression of Inducible NO Synthase in Vascular Smooth Muscle Cells During Aging Is Associated With Enhanced NF-κB Activation
Authors: Yan Zhong-qun , Sirsjö Allan , Bochaton-Piallat Marie-Luce , Gabbiani Giulio , Hansson Göran K.
Published: June 2011

Abstract—Vascular smooth muscle cells (SMCs) are important targets for endothelium-derived nitric oxide (NO), but this production is attenuated in injured and diseased arteries and during aging. However, SMCs can produce NO themselves by expressing an inducible form of NO synthase (iNOS) under inflammatory conditions and in the repair process after arterial injury. We examined iNOS expression in SMCs derived from the aortic media of newborn, young adult, and old rats. Our results show that SMCs from newborn rats cannot produce significant amounts of NO on stimulation with interferon-γ plus lipopolysaccharide or interleukin-1β. In contrast, SMCs from old rats exhibit markedly enhanced iNOS activity. The difference in iNOS activity between the newborn and the old SMCs was closely correlated with levels of iNOS protein, mRNA, and gene promoter activity. Similarly, intercellular adhesion molecule-1 (ICAM-1) was also expressed more abundantly in the old than in the newborn SMCs in response to cytokines. Both iNOS and ICAM-1 are transcriptionally regulated by nuclear factor κB (NF-κB). Our data demonstrate an intense transactivation of NF-κB in old SMCs on tumor necrosis factor-α stimulation but only a weak one in newborn SMCs. The difference in the NF-κB activation could be explained by a much faster and more extensive IκBα degradation in old than in newborn SMCs. These data indicate that the capability to respond to proinflammatory stimuli by activating NF-κB differs between SMCs at different stages of development. This results in differential capability to express NF-κB–dependent genes such as iNOS and ICAM-1, which could have implications for host defense and the pathogenesis of vascular diseases.
Accelerated Neointima Formation After Vascular Injury in Mice With Stromelysin-3 (MMP-11) Gene Inactivation
Authors: Lijnen H. Roger , Van Hoef Berthe , Vanlinthout Ingrid , Verstreken Maria , Rio Marie-Christine , Collen Désiré
Published: June 2011

Abstract
—The hypothesis that stromelysin-3 (MMP-11), a unique member of the matrix metalloproteinase (MMP) family, plays a role in neointima formation was tested with the use of a vascular injury model in wild-type (MMP-11
+/+
) and MMP-11–deficient (MMP-11
−/−
) mice. Neointima formation 2 to 3 weeks after electric injury of the femoral artery was significantly enhanced in MMP-11
−/−
as compared with MMP-11
+/+
mice, in both mice of a pure 129SV genetic background (0.014 versus 0.0010 mm
2
at 2 weeks,
P
<0.001) and those of a 50/50 mixed 129SV/BL6 background (0.030 versus 0.013 mm
2
at 3 weeks,
P
<0.05). The medial areas were comparable, resulting in intima/media ratios that were significantly increased in MMP-11
−/−
as compared with MMP-11
+/+
arteries, in mice of both the 129SV (1.0 versus 0.18,
P
<0.001) and mixed (1.5 versus 0.70,
P
<0.05) backgrounds. Nuclear cell counts in cross-sectional areas of the intima of the injured region were higher in arteries from MMP-11
−/−
mice than in those from MMP-11
+/+
mice (210 versus 48,
P
<0.001, in pure 129SV mice and 290 versus 150,
P
<0.01, in mice of the mixed genetic background). Immunocytochemical analysis revealed that α-actin–positive and CD45-positive cells were more abundant in intimal sections of MMP-11
−/−
mice. Degradation of the internal elastic lamina was more extensive in arteries of MMP-11
−/−
mice than in those of MMP-11
+/+
mice (39% versus 6.8% at 3 weeks,
P
<0.005). The mechanisms by which MMP-11 could impair elastin degradation and cellular migration in this model remain, however, unknown.
Subendothelial Cells From Normal Bovine Arteries Exhibit Autonomous Growth and Constitutively Activated Intracellular Signaling
Authors: Frid Maria G. , Aldashev Almaz A. , Nemenoff Raphael A. , Higashito Ryuji , Westcott Jay Y. , Stenmark Kurt R.
Published: June 2011

Abstract
—The arterial media is comprised of heterogeneous smooth muscle cell (SMC) subpopulations with markedly different growth responses to pathophysiological stimuli. Little information exists regarding the intracellular signaling pathways that contribute to these differences. Therefore, we investigated the growth-related signaling pathways in a unique subset of subendothelial SMCs (L1 cells) from normal, mature, bovine arteries and compared them with those in “traditional” SMCs derived from the middle media (L2 SMCs). Subendothelial L1 cells exhibited serum-independent autonomous growth, not observed in L2 SMCs. Autonomous growth of L1 cells was driven largely by the constitutively activated extracellular signal–regulated kinase (ERK-1/2) cascade. Inhibition of upstream activators of ERKs (MAP kinase kinase-1, p21
ras
, receptor tyrosine kinases, and Gi protein–coupled receptors) led to suppression of autonomous growth in these cells. L1 cells also exhibited constitutive activation of important downstream targets of ERKs (cytosolic phospholipase A
2
, cyclooxygenase-2) and secreted large amounts of prostaglandins. Importantly, L1 cells secreted potent mitogenic factor(s), which could potentially contribute in an autocrine fashion to the constitutive activation of these cells. Our data suggest that unique arterial cells with autonomous growth potential and constitutively activated signaling pathways exist in normal arteries and may contribute selectively to the pathogenesis of vascular diseases.
Lipoprotein-Associated Phospholipase A
2
, Platelet-Activating Factor Acetylhydrolase, Is Expressed by Macrophages in Human and Rabbit Atherosclerotic Lesions
Authors: Häkkinen Tomi , Luoma Jukka S. , Hiltunen Mikko O. , Macphee Colin H. , Milliner Kevin J. , Patel Lisa , Rice Simon Q. , Tew David G. , Karkola Kari , Ylä-Herttuala Seppo
Published: June 2011

Abstract
—We studied the expression of lipoprotein-associated phospholipase A
2
(Lp-PLA
2
), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA
2
activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA
2
mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase—polymerase chain reaction analysis indicated an increased expression of Lp-PLA
2
mRNA in human atherosclerotic lesions. In addition, ≈6-fold higher Lp-PLA
2
activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA
2
, which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA
2
activity could lead to antiatherogenic effects in the vessel wall.
Altered Vascular Injury Responses in Mice Deficient in Protease-Activated Receptor-1
Authors: Cheung Wai-man , D’Andrea Michael R. , Andrade-Gordon Patricia , Damiano Bruce P.
Published: June 2011

Abstract
—Expression of protease-activated receptor-1 (PAR-1), a cell-surface receptor for thrombin, is increased in balloon-injured rat carotid artery and human atherosclerotic tissue. To examine the role of PAR-1 in vascular injury, we compared vascular injury responses in wild-type (WT) and PAR-1–deficient (PAR-1
−/−
) mice. Arterial injury was induced by inserting a flexible guidewire into the common carotid artery and withdrawing it 6 times with rotation. Bromodeoxyuridine, delivered subcutaneously by osmotic minipump, was used to measure cellular proliferation. Mice were perfusion-fixed at 1, 2, 5, 10, and 14 days after injury. Extensive endothelial damage, mural thrombosis, platelet adherence, and medial smooth muscle cell loss and necrosis were apparent at day 1 in both WT and PAR-1
−/−
mice. The incidence of thrombosis or platelet deposition in WT and PAR-1
−/−
mice declined from 100% at day 1 to 25% and 21%, respectively, at 14 days. Endothelial disruption, as assessed by Evan’s blue uptake, was maximum at day 1 and declined by day 14. This apparent endothelial regrowth was similar in WT and PAR-1
−/−
mice. Significant medial thickening at 14 days after injury was similar in WT (from 22.8±1.7 to 30.7±1.9 μm) and PAR-1
−/−
(from 23.2±2.1 to 30.5±2.2 μm) mice. Medial area also increased in response to injury but to a lesser extent in PAR-1
−/−
mice (from 0.0250±0.0044 to 0.0312±0.0047 mm
2
) than in WT mice (from 0.0266±0.0040 to 0.0398±0.0050 mm
2
). Neointima was variable and occurred in 6 of 13 WT and 5 of 12 PAR-1
−/−
mice. However, intimal area tended to be less in PAR-1
−/−
mice (0.0016±0.0007 mm
2
) compared with WT mice (0.0082±0.0032 mm
2
), although this difference did not achieve statistical significance (
P
=0.06). Cell density was significantly greater in normal carotids from PAR-1
−/−
(6.4±0.5×10
3
/mm
2
) compared with WT (4.3±0.8×10
3
/mm
2
) mice and remained elevated after injury. Vessel and lumen diameters tended to increase in WT mice after injury, whereas vessel diameter was unchanged and lumen diameter actually decreased in PAR-1
−/−
mice. Cell proliferation in injured carotid arteries was similar in PAR-1
−/−
and WT mice. These data suggest that PAR-1
−/−
may play a role in vascular injury responses in this mouse model via possible effects on extracellular matrix regulation.