bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma Leptin and the Risk of Cardiovascular Disease in the West of Scotland Coronary Prevention Study (WOSCOPS)
Authors: Wallace A. Michael , McMahon Alex D. , Packard Chris J. , Kelly Anne , Shepherd James , Gaw Allan , Sattar Naveed
Published: September 2007

Background
—

Leptin plays a role in fat metabolism and correlates with insulin resistance and other markers of the metabolic syndrome, independent of total adiposity. Therefore, we hypothesized that raised leptin levels may identify men at increased risk of a coronary event in the West of Scotland Coronary Prevention Study (WOSCOPS).

Methods and Results
—

Plasma leptin levels were measured at baseline in 377 men (cases) who subsequently experienced a coronary event and in 783 men (controls) who remained free of an event during the 5-year follow-up period of the study. Controls were matched to cases on the basis of age and smoking history and were representative of the entire WOSCOPS cohort. Leptin levels were significantly higher in cases than controls (5.87±2.04 ng/mL versus 5.04±2.09 ng/mL,
P
<0.001). In univariate analysis, for each 1 SD increase in leptin, the relative risk (RR) of an event increased by 1.25 (95% confidence interval [CI], 1.10 to 1.43;
P
<0.001). There was minimal change in this RR with correction for body mass index (RR, 1.24; 95% CI, 1.06 to 1.45;
P
=0.006) or with further correction for classic risk factors, including age, lipids, and systolic blood pressure (RR, 1.20; 95% CI, 1.02 to 1.42;
P
=0.03). Leptin correlated with C-reactive protein (
r
=0.24,
P
<0.001) and, even with this variable added to the model, leptin retained significance as a predictor of coronary events (RR, 1.18; 95% CI, 1.00 to 1.39;
P
=0.05) at the expense of C-reactive protein.

Conclusions
—

We show, for the first time, in a large prospective study that leptin is a novel, independent risk factor for coronary heart disease.
Novel PRKAG2 Mutation Responsible for the Genetic Syndrome of Ventricular Preexcitation and Conduction System Disease With Childhood Onset and Absence of Cardiac Hypertrophy
Authors: Gollob Michael H. , Seger John J. , Gollob Tanya N. , Tapscott Terry , Gonzales Oscar , Bachinski Linda , Roberts Robert
Published: September 2007

Background
—

We recently reported a mutation in the PRKAG2 gene to be responsible for a familial syndrome of ventricular preexcitation, atrial fibrillation, conduction defects, and cardiac hypertrophy. We now report a novel mutation in PRKAG2 causing Wolff-Parkinson-White syndrome and conduction system disease with onset in childhood and the absence of cardiac hypertrophy.

Methods and Results
—

DNA was extracted from white blood cells obtained from family members. PRKAG2 exons were amplified by polymerase chain reaction and were screened for mutations by direct sequencing. The genomic organization of the PRKAG2 gene was determined using inter-exon long-range polymerase chain reaction for cDNA sequence not available in the genome database. A missense mutation, Arg531Gly, was identified in all affected individuals but was absent in 150 unrelated individuals. The PRKAG2 gene was determined to consist of 16 exons and is at least 280 kb in size.

Conclusions
—

We identified a novel mutation (Arg531Gly) in the γ-2 regulatory subunit (PRKAG2) of AMP-activated protein kinase (AMPK) to be responsible for a syndrome associated with ventricular preexcitation and early onset of atrial fibrillation and conduction disease. These observations confirm an important functional role of AMPK in the regulation of ion channels specific to cardiac tissue. The identification of the cardiac ion channel(s) serving as substrate for AMPK not only would provide insight into the molecular basis of atrial fibrillation and heart block but also may suggest targets for the development of more specific therapy for these common rhythm disturbances.
Ischemic Preconditioning Upregulates Vascular Endothelial Growth Factor mRNA Expression and Neovascularization via Nuclear Translocation of Protein Kinase C ε in the Rat Ischemic Myocardium
Authors: Kawata Hiroyuki , Yoshida Ken-ichi , Kawamoto Atsuhiko , Kurioka Hideyuki , Takase Eiji , Sasaki Yasunobu , Hatanaka Kazuhito , Kobayashi Masahiko , Ueyama Takashi , Hashimoto Toshio , Dohi Kazuhiro
Published: September 2007

Abstract
—Ischemic preconditioning (IP) exerts cardioprotection through protein kinase C (PKC) activation, whereas myocardial ischemia enhances vascular endothelial growth factor (VEGF) mRNA expression. However, the IP effect or the involvement of PKC on the VEGF expression is unknown in myocardial infarction. We investigated whether IP enhances VEGF gene expression and angiogenesis through PKC activation in the in vivo myocardial infarction model. Sprague-Dawley rats were assigned into the following 3 groups: the sham group; the IP group, which underwent 3 cycles of 3 minutes of ischemia and 5 minutes of reperfusion (IP procedure); and the non-IP group. The latter 2 groups were subsequently subjected to left anterior descending coronary artery occlusion. To examine the involvement of PKC, the PKC inhibitor chelerythrine (5 mg/kg) or bisindolylmaleimide (1 mg/kg) was injected intravenously before the IP procedures. PKCε was translocated to the nucleus after 10 minutes of ischemia after the IP procedure but was not translocated in the non-IP and the sham groups. VEGF mRNA expression 3 hours after infarction was significantly higher in the IP group than in the non-IP and the sham groups. Capillary density in the infarction was significantly higher, whereas the infarct size was smaller in the IP group than in the non-IP group at 3 days of infarction. Chelerythrine but not bisindolylmaleimide blocked all of the IP effects on the nuclear translocation of PKCε, enhancement of VEGF mRNA expression and angiogenesis, and infarct size limitation. These results show that IP may enhance VEGF gene expression and angiogenesis through nuclear translocation of PKCε in the infarcted myocardium.
Myocardial Akt Activation and Gender
Authors: Camper-Kirby Dreama , Welch Sara , Walker Angela , Shiraishi Isao , Setchell Kenneth D. R. , Schaefer Erik , Kajstura Jan , Anversa Piero , Sussman Mark A.
Published: September 2007

Abstract
—Cardiovascular disease risk is higher in men than women, but the basis for this discrepancy remains controversial. Estrogenic stimulation of the myocardium or isolated cardiomyocytes has been purported to exert multiple beneficial effects associated with inhibition of maladaptive responses to pathogenic insults. This report describes a significant difference between the sexes in myocardial activation of Akt, a protein kinase that regulates a broad range of physiological responses including metabolism, gene transcription, and cell survival. We find that young women possess higher levels of nuclear-localized phospho-Akt
473
relative to comparably aged men or postmenopausal women. Both localization of phospho-Akt
473
in myocardial nuclei of sexually mature female mice versus males and Akt kinase activity in nuclear extracts of hearts from female mice versus males are elevated. Cytosolic localization of phospho-forkhead, a downstream nuclear target of Akt, is also increased in female relative to male mice, suggesting a potential mechanism for cardioprotective nuclear signaling resulting from Akt activation. Phospho-Akt
473
levels and localization at cardiac nuclei are similarly increased in transgenic mice with myocardium-specific expression of insulin-like growth factor I, a proven stimulus for Akt activation. Phospho-Akt
473
is also localized to the nucleus of cultured cardiomyocytes after exposure to 17β-estradiol or genistein (a phytoestrogen in soy protein–based diets), and neonatal exposure of litters to genistein elevated nuclear phospho-Akt
473
localization. The activation of Akt in a gender-dependent manner may help explain differences observed in cardiovascular disease risk between the sexes and supports the potential beneficial effects of estrogenic stimulation.
Potential Ionic Mechanism for Repolarization Differences Between Canine Right and Left Atrium
Authors: Li Danshi , Zhang Liming , Kneller James , Nattel Stanley
Published: September 2007

Abstract
—Experimental and clinical evidence suggests a critical role for the left atrium (LA) in atrial fibrillation (AF). In animal models, repolarization is faster in the LA than in the right atrium (RA), leading to more stable reentry circuits with a shorter intrinsic period in the LA. The ionic mechanisms underlying LA-RA repolarization differences are unknown. Therefore, we evaluated ionic currents and action potentials (APs) with the whole-cell patch clamp in isolated canine atrial myocytes. The density of the rapid delayed rectifier current (
I
Kr
) was greater in the LA (eg, 1.83±0.10 pA/pF at +20 mV) than in the RA (1.15±0.07 pA/pF,
P
<0.01; n=16 cells per group). The slow and ultrarapid delayed rectifier, the inward rectifier, L-type Ca
2+
, and transient outward K
+
currents were all comparable in the LA and RA. There were no differences in kinetic or voltage-dependent properties of currents in LA versus RA. Western blots of ether-a-go-go–related gene (ERG) protein in three RA and corresponding LA regions showed significantly greater ERG expression in LA. AP duration (APD) was shorter in the LA versus RA in both isolated cells and multicellular preparations, and the effective refractory period (ERP) was shorter in the LA compared with the RA in vivo. Dofetilide had significantly larger APD- and ERP-increasing effects in the LA compared with RA, and LA-RA repolarization differences were eliminated by exposure to dofetilide. We conclude that LA myocytes have larger
I
Kr
than do RA myocytes, contributing importantly to the shorter APD and ERP in LA. The larger LA
I
Kr
may participate in the ability of the LA to act as a “driver region” for AF, with potentially important implications for understanding AF mechanisms and antiarrhythmic therapy.
Cardiovascular Defects Associated With Abnormalities in Midline Development in the
Loop-tail
Mouse Mutant
Authors: Henderson Deborah J. , Conway Simon J. , Greene Nicholas D. E. , Gerrelli Dianne , Murdoch Jennifer N. , Anderson Robert H. , Copp Andrew J.
Published: September 2007

Abstract
—
Loop-tail
(
Lp
) is a naturally occurring mouse mutant that develops severe neural tube defects. In this study, we describe complex cardiovascular defects in
Lp
homozygotes, which include double-outlet right ventricle, with obligatory perimembranous ventricular septal defects, and double-sided aortic arch, with associated abnormalities in the aortic arch arteries. Outflow tract and aortic arch defects are often related to abnormalities in the cardiac neural crest, but using molecular and anatomic markers, we show that neural crest migration is normal in
Lp/Lp
embryos. On the other hand, the heart fails to loop normally in
Lp/Lp
embryos, in association with incomplete axial rotation and reduced cervical flexion. As a consequence, the ventricular loop is shifted posteromedially relative to its position in wild-type embryos. This suggests that the observed cardiac alignment defects in the
Lp
mutant may be secondary to failure of neural tube closure and incomplete axial rotation. Double-sided aortic arch is a rare finding among mouse models. In humans, it is usually an isolated malformation, only rarely occurring in combination with other cardiac defects. We suggest that the double-sided arch arises as a primary defect in the
Lp
mutant, unrelated to the alignment defects, perhaps reflecting a role for the (as-yet-unknown)
Lp
gene in maintenance/regression of the aortic arch system.
Vascular Smooth Muscle Cell–Directed Overexpression of Heme Oxygenase-1 Elevates Blood Pressure Through Attenuation of Nitric Oxide–Induced Vasodilation in Mice
Authors: Imai Tomihiko , Morita Toshisuke , Shindo Takayuki , Nagai Ryozo , Yazaki Yoshio , Kurihara Hiroki , Suematsu Makoto , Katayama Shigehiro
Published: September 2007

Abstract
—To elucidate pathophysiological roles of heme oxygenase (HO)-1 in regulation of vascular tone in vivo, we have developed and characterized transgenic (Tg) mice that overexpress HO-1 site specifically in vascular smooth muscle cells (VSMCs). The Tg mice were generated by use of human HO-1 cDNA under the control of SM22-α promoter. The HO-1 gene overexpression was demonstrated by Northern blot analysis and coincided with increases in the protein expression in VSMCs and total HO activities. Tg mice exhibited a significant increase in arterial pressure at various ages and displayed impaired nitrovasodilatory responses in isolated aortic segments versus nontransgenic littermates while enhancing their nitric oxide (NO) production. The pressure of Tg mice was unchanged by systemic administration of either
N
ω
-nitro-
l
-arginine or SNP. Furthermore, the isolated aorta in these mice exhibited lesser extents of NO-elicited cGMP elevation via soluble guanylate cyclase (sGC), while exhibiting no notable downregulation of sGC expression. Such impairment of the NO-elicited cGMP increase was restored significantly by tin protoporphyrin IX, an HO inhibitor. On the other hand, 3-(5′-hydroxymethyl-2′ furyl)-1-benzyl-indazol (YC-1), an NO-independent activator of sGC, increased cGMP and relaxed aortas from Tg mice to levels comparable with those from nontransgenic mice, which indicates that contents of functionally intact sGC are unlikely to differ between the two systems. These findings suggest that site-specific overexpression of HO-1 in VSMCs suppresses vasodilatory response to NO and thereby leads to an elevation of arterial pressure.
Phenotypic Modulation of Vascular Smooth Muscle Cells Induced by Unsaturated Lysophosphatidic Acids
Authors: Hayashi Ken’ichiro , Takahashi Masanori , Nishida Wataru , Yoshida Kenji , Ohkawa Yasuyuki , Kitabatake Akira , Aoki Junken , Arai Hiroyuki , Sobue Kenji
Published: September 2007

The phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state to the dedifferentiated one is critically involved in the development and progression of atherosclerosis. Although many cytokines and growth factors have been reported as atherogenic factors, the critical pathogens for inducing atherosclerosis remain unknown, largely because proper examining systems of them have not been developed. We recently established primary culture systems for visceral SMCs and VSMCs in which both SMCs, when cultured on laminin with insulin-like growth factor-I, show a differentiated phenotype, as indicated by a spindle-like shape, ligand-induced contractility, and a high level of SMC differentiation marker gene expression. In this study, we searched for critical dedifferentiation factors for these SMCs using our culture system. We found that polar lipids extracted from human serum markedly induced VSMC dedifferentiation, and this activity was solely present in the lysophosphatidic acid (LPA) fraction. Among several LPA species detected in human serum lipids, unsaturated LPAs were identified as major contributors to the induction of VSMC dedifferentiation. Signaling and phenotype analyses revealed that unsaturated LPA–induced VSMC dedifferentiation is mediated through the coordinated activation of extracellular signal–regulated kinase and p38 mitogen–activated protein kinase. Thus, this report demonstrates the first finding that unsaturated LPAs, but not saturated LPAs, specifically induce VSMC phenotypic modulation, suggesting that these molecules could function as atherogenic factors.
Antiinflammatory and Antiarteriosclerotic Actions of HMG-CoA Reductase Inhibitors in a Rat Model of Chronic Inhibition of Nitric Oxide Synthesis
Authors: Ni Weihua , Egashira Kensuke , Kataoka Chu , Kitamoto Shiro , Koyanagi Masamichi , Inoue Shujiro , Takeshita Akira
Published: September 2007

Recent studies suggest that some of the beneficial effects of 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) may be due to their cholesterol-lowering independent effects on the blood vessels. Chronic inhibition of endothelial nitric oxide (NO) synthesis by oral administration of

N
ω

-nitro-
l
-arginine methyl ester (L-NAME) to rats induces early vascular inflammation as well as subsequent arteriosclerosis. The aim of the study is to test whether treatment with statins attenuates such arteriosclerotic changes through their cholesterol-lowering independent effects. We investigated the effect of statins (pravastatin and cerivastatin) on the arteriosclerotic changes in the rat model. We found that treatment with statins did not affect serum lipid levels but markedly inhibited the L-NAME–induced vascular inflammation and arteriosclerosis. Treatment with statins augmented endothelial NO synthase activity in L-NAME–treated rats. We also found the L-NAME induced increase in Rho membrane translocation in hearts and its prevention by statins. Such vasculoprotective effects of statins were suppressed by the higher dose of L-NAME. In summary, in this study, we found that statins such as pravastatin and cerivastatin inhibited vascular inflammation and arteriosclerosis through their lipid-lowering independent actions in this model. Such antiarteriosclerotic effects may involve the increase in endothelial NO synthase activity and the inhibition of Rho activity.
Reactive Oxygen Species Mediate Amplitude-Dependent Hypertrophic and Apoptotic Responses to Mechanical Stretch in Cardiac Myocytes
Authors: Pimentel David R. , Amin Jay K. , Xiao Lei , Miller Thomas , Viereck Jason , Oliver-Krasinski Jennifer , Baliga Ragavendra , Wang Jing , Siwik Deborah A. , Singh Krishna , Pagano Patrick , Colucci Wilson S. , Sawyer Douglas B.
Published: September 2007

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated whether oxidative stress mediates hypertrophy and apoptosis in cyclically stretched ventricular myocytes. Neonatal rat ventricular myocytes cultured on laminin-coated silastic membranes were stretched cyclically (1 Hz) at low (nominal 5%) and high (nominal 25%) amplitudes for 24 hours. Stretch caused a graded increase in superoxide anion production as assessed by superoxide dismutase (SOD)-inhibitable cytochrome
c
reduction or electron paramagnetic resonance spectroscopy. The role of reactive oxygen species (ROS) was assessed using the cell-permeable SOD/catalase mimetics Mn(II/III)tetrakis(1-methyl-4-peridyl) (MnTMPyP) and EUK-8. Stretch-induced increases in protein synthesis (
3
H-leucine incorporation) and cellular protein content were completely inhibited by MnTMPyP (0.05 mmol/L) at both low and high amplitudes of stretch. In contrast, while MnTMPyP inhibited basal atrial natriuretic factor (ANF) mRNA expression, the stretch-induced increase in ANF mRNA expression was not inhibited by MnTMPyP. In contrast to hypertrophy, only high-amplitude stretch increased myocyte apoptosis, as reflected by increased DNA fragmentation on gel electrophoresis and an ≈3-fold increase in the number of TUNEL-positive myocytes. Similarly, only high-amplitude stretch increased the expression of bax mRNA. Myocyte apoptosis and bax expression stimulated by high-amplitude stretch were inhibited by MnTMPyP. Both low- and high-amplitude stretch caused rapid phosphorylation of ERK1/2, while high-, but not low-, amplitude stretch caused phosphorylation of JNKs. Activation of both ERK1/2 and JNKs was ROS-dependent. Thus, cyclic strain causes an amplitude-related increase in ROS, associated with differential activation of kinases and induction of hypertrophic and apoptotic phenotypes.
Targeted Overexpression of CCAAT/Enhancer-Binding Protein-δ Evokes Enhanced Gene Transcription of Platelet-Derived Growth Factor α-Receptor in Vascular Smooth Muscle Cells
Authors: Yang Zhao-Hui , Kitami Yutaka , Takata Yasunori , Okura Takafumi , Hiwada Kunio
Published: September 2007

Abstract—

Platelet-derived growth factor (PDGF) is thought to play a significant role in various models of vascular remodeling, particularly in the early process of vascular diseases. Its action is mediated by its specific receptor, the PDGF receptor. The PDGF α-receptor (PDGFαR) plays an important role in the growth and proliferation of vascular smooth muscle cells (VSMCs), and its gene expression is thought to be regulated by several potential transcriptional nuclear factors. However, the detailed mechanisms of tissue-specific transactivation of the PDGFαR gene in VSMCs remain to be clarified. We have previously demonstrated that the rat PDGFαR gene contains an enhancer core sequence for CCAAT/enhancer-binding proteins (C/EBPs) in its promoter region, and we have also suggested that C/EBP-δ is the principal factor involved in the induction of tissue-specific transcriptional activity of the PDGFαR gene in VSMCs. To explore the definitive roles of C/EBP-δ protein on PDGFαR gene transcription in VSMCs, we developed C/EBP-δ transgenic rats by using a chimeric fusion gene of the mouse smooth muscle α-actin promoter and an entire coding region of rat C/EBP-δ cDNA. This report describes the first successful targeted overexpression of C/EBP-δ capable of inducing PDGFαR gene transcription and modifying cell proliferative activity to PDGFs. Targeted overexpression of C/EBP-δ evokes high levels of PDGFαR gene expression, susceptibility to VSMC growth, and proliferation of VSMCs to PDGFs. The results obtained reveal evidence of a new role and new functional significance of C/EBP-δ on VSMC growth via the PDGFαR during the process of vascular remodeling and atherosclerosis.
Increased Expression and Activity of RhoA Are Associated With Increased DNA Synthesis and Reduced p27
Kip1
Expression in the Vasculature of Hypertensive Rats
Authors: Seasholtz Tammy M. , Zhang Tong , Morissette Michael R. , Howes Amy L. , Yang Amy H. , Brown Joan Heller
Published: September 2007

Abstract—

We have previously shown that the function of the small G protein Rho is required for vascular smooth muscle cell proliferation and migration. We hypothesized that changes in Rho or Rho signaling might contribute to enhanced vascular proliferative responses associated with hypertension. Western blot analysis revealed that total RhoA expression was ≈2-fold higher in aortas, tail arteries, and aortic smooth muscle cells (ASMCs) obtained from adult male spontaneously hypertensive rats (SHR) compared with those from Wistar Kyoto rats (WKY). An increase in active GTP-bound RhoA was detected in aortic homogenates by affinity precipitation with the RhoA effector rhotekin and by examining RhoA-[
35
S]GTPγS binding. RhoA protein and activity were also increased in vessels from rats treated with
N
-nitro-
l
-arginine methyl ester to increase blood pressure. Thrombin-stimulated RhoA activation was also significantly greater in ASMCs from SHR. As a functional correlate of these changes in Rho signaling, thrombin-stimulated DNA synthesis was enhanced in tail arteries and ASMCs from SHR. Expression of the cyclin-dependent kinase inhibitor p27
Kip1
was decreased by two thirds in SHR, and this decrease was mimicked in ASMCs by expression of a constitutively active (GTPase-deficient) mutant of RhoA. Wortmannin (10 nmol/L) fully inhibited the decrease in p27
Kip1
induced by RhoA, and a membrane-targeted catalytic subunit of phosphatidylinositol-3 kinase (PI3K [p110
CAAX
]) decreased p27
Kip1
expression, suggesting that RhoA signals through PI3K. These data provide evidence that RhoA brings about changes in DNA synthesis through reduced expression of p27
Kip1
, mediated in part via PI3K, and suggest that increases in RhoA expression and activity contribute to the enhanced vascular responsiveness observed in hypertension.
Novel Clinical Markers of Vascular Wall Inflammation
Authors: Blake Gavin J. , Ridker Paul M.
Published: September 2007

Inflammatory processes play a pivotal role in the pathogenesis of atherosclerosis and mediate many of the stages of atheroma development from initial leukocyte recruitment to eventual rupture of the unstable atherosclerotic plaque. Elevated plasma levels of several markers of the inflammatory cascade have been shown to predict future risk of plaque rupture. These markers include P-selectin, interleukin-6, tumor necrosis factor-α, soluble intercellular adhesion molecule-1, and C-reactive protein (CRP). Produced in the liver in response to interleukin-6, CRP has emerged as the most powerful inflammatory marker of future cardiovascular risk. Initially considered an innocent bystander in the atherosclerotic process, recent evidence suggests that CRP may have direct proinflammatory effects. Numerous large-scale, prospective studies have found that elevated baseline levels of CRP are a strong independent predictor of future vascular risk. Furthermore, aspirin and statin therapy appear to be particularly effective among individuals with high CRP levels. The addition of CRP screening to traditional lipid testing has the potential to identify individuals at high risk for future cardiovascular events who may benefit from targeted preventive interventions.
p38 Triggers Late Preconditioning Elicited by Anisomycin in Heart
Authors: Zhao Ting C. , Taher Mohiuddin M. , Valerie Kristoffer C. , Kukreja Rakesh C.
Published: September 2007

We investigated the role of stress-activated p38 MAP kinase (p38/SAPK-2) signaling in delayed preconditioning of the heart. Adult male out-bred ICR mice were treated with p38 activator, anisomycin (0.1 mg/kg IP), or vehicle (5% DMSO). Twenty-four hours later, hearts were perfused in Langendorff mode and subjected to 30 minutes of ischemia and 30 minutes of reperfusion. Improvement in postischemic recovery of end-diastolic pressure and reduction in infarct size was observed, which was abolished by SB203580, a specific p38 inhibitor, and pyrrolidinediethyldithiocarbamate (PDTC), the NF-κB inhibitor, but not by PD 98059, a specific inhibitor for MEK1 or 2. Transient increase in p38 phosphorylation was observed 15 minutes after anisomycin treatment which subsided by 30 minutes. Electrophoretic mobility shift assay demonstrated rapid activation of NF-κB DNA binding with anisomycin, peaking at 30 minutes. Western blot confirmed the accumulation of p50 and p65 in nuclear extracts after anisomycin treatment. Anisomycin-induced NF-κB DNA binding activity was inhibited by SB203580 and PDTC. Expression of inducible nitric oxide synthase (iNOS) mRNA, protein, and nitric oxide (NO) synthesis were enhanced in anisomycin-treated mice. SB203580 and PDTC blocked the increased expression of iNOS and increase in synthesis of NO. Selective iNOS inhibitor
S
-methylisothiourea abolished the protective effect of anisomycin. Furthermore, postischemic cardioprotective effect of anisomycin was absent in mice with targeted ablation of iNOS gene but not in the wild-type B6.129 mice. For the first time, these results suggest that direct pharmacological activation of p38 triggers delayed preconditioning by signaling mechanism involving NF-κB activation and synthesis of NO from iNOS.
Cytomegalovirus Infection Accelerates Inflammation in Vascular Tissue Overexpressing Monocyte Chemoattractant Protein-1
Authors: Froberg M. Kent , Adams Alice , Seacotte Nicole , Parker-Thornburg Jan , Kolattukudy Pappachan
Published: September 2007

Cardiovascular disease is the leading cause of mortality in the United States. Atherosclerosis is responsible for most of this pathology and is an inflammatory disease with multiple cytokines and adhesion molecules expressed during atherogenesis. Cytomegalovirus (CMV), monocytes, and monocyte chemoattractant protein-1 (MCP-1) have all been implicated in human atherogenesis. A transgenic mouse overexpressing MCP-1 in the myocardium and pulmonary arteries develops myocarditis and pulmonary vascular inflammation. We infected MCP-1 transgenic mice with a sublethal dose of murine cytomegalovirus (MCMV) to look for evidence of accelerated inflammation in vascular tissues overexpressing MCP-1 to determine if MCMV could interact with monocytes and MCP-1 in a manner similar to what may occur in atherogenesis. MCMV infection of MCP-1 transgenic mice caused ascites, myocarditis, and pulmonary artery inflammation, which was not present in mock-infected MCP-1 or MCMV-infected wild-type mice. Inflammatory infiltrates in these tissues consisted of macrophages and T lymphocytes similar to the infiltrates seen in atherosclerosis. Virus presence in inflamed tissues was demonstrated by infecting transgenic mice with MCMV recombinant virus containing the gene sequence for the enhanced green fluorescent protein (EGFP). Human CMV could be involved in atherogenesis in a similar manner by interacting with monocytes and MCP-1 specifically expressed in vascular walls.
Signaling Mechanisms of Nuclear Factor-κB-Mediated Activation of Inflammatory Genes by 13-Hydroperoxyoctadecadienoic Acid in Cultured Vascular Smooth Muscle Cells
Authors: Natarajan Rama , Reddy Marpadga A. , Malik Kafait U. , Fatima Soghra , Khan Bobby V.
Published: September 2007

Oxidatively modified low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. LDL oxidation may be mediated by several factors, including cellular lipoxygenases. The lipoxygenase product of linoleic acid, 13-hydroperoxyoctadecadienoic acid (13-HPODE), is a significant component of oxidized LDL and has been shown to be present in atherosclerotic lesions. However, the mechanism of action of these oxidized lipids in vascular smooth muscle cells (VSMCs) is not clear. In the present study, we show that 13-HPODE leads to the activation of Ras as well as the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2, p38, and c-Jun amino-terminal kinase, in porcine VSMCs. 13-HPODE also specifically activated the oxidant stress-responsive transcription factor, nuclear factor-κB, but not activator protein-1 or activator protein-2. 13-HPODE-induced nuclear factor-κB DNA binding activity was blocked by an antioxidant,
N
-acetylcysteine, as well as an inhibitor of protein kinase C. 13-HPODE, but not the hydroxy product, 13-(
S
)-hydroxyoctadecadienoic acid, also dose-dependently increased vascular cell adhesion molecule-1 promoter activation. This was inhibited by an antioxidant as well as by inhibitors of Ras p38 mitogen-activated protein kinase and protein kinase C. Our results suggest that oxidized lipid components of oxidized LDL, such as 13-HPODE, may play a key role in the atherogenic process by inducing the transcriptional regulation of inflammatory genes in VSMCs via the activation of key signaling kinases.
Angiotensin II Administration to Atherosclerotic Mice Increases Macrophage Uptake of Oxidized LDL
Authors: Keidar Shlomo , Heinrich Ronit , Kaplan Marielle , Hayek Tony , Aviram Michael
Published: September 2007

The goal of the present study was to elucidate mechanisms for angiotensin II (Ang II) induction of oxidized low density lipoprotein (Ox-LDL) uptake by macrophages, the hallmark of early atherosclerosis. Compared with placebo treatment, Ang II injections (0.1 mL, 10
−7
mol/L per day) for 2 weeks to apolipoprotein E-deficient mice significantly increased Ox-LDL degradation, CD36 mRNA expression, and CD36 protein expression by their peritoneal macrophages (MPMs). These effects were abolished by treatment with losartan (5 to 50 mg/kg per day) before Ang II administration. Because no such effect was obtained in vitro, the ex vivo effect of Ang II on macrophage uptake of Ox-LDL could be mediated by a factor that is not expressed at a significant level in vitro. Because Ang II stimulates cellular production of interleukin-6 (IL-6), we analyzed the possible role of IL-6 as a mediator of Ang II-mediated cellular uptake of Ox-LDL by using several approaches. First, incubations of IL-6 with MPM or IL-6 administration in mice increased macrophage Ox-LDL degradation and CD36 mRNA expression. Second, injection of IL-6 receptor antibodies in mice during Ang II treatment reduced macrophage Ox-LDL uptake and CD36 expression compared treatment with Ang II alone. Finally, Ang II treatment of IL-6-deficient mice did not affect their MPM Ox-LDL uptake and CD36 protein levels. Thus, we conclude that a novel mechanism for Ang II atherogenicity, related to macrophage cholesterol accumulation and foam cell formation, may involve its stimulatory effect on macrophage uptake of Ox-LDL, a process mediated byIL-6.
Expression of Glutaredoxin in Human Coronary Arteries
Authors: Okuda Masanori , Inoue Nobutaka , Azumi Hiroshi , Seno Tadashi , Sumi Yoshihiko , Hirata Ken-ichi , Kawashima Seinosuke , Hayashi Yoshitake , Itoh Hiroshi , Yodoi Junji , Yokoyama Mitsuhiro
Published: September 2007

Oxidative stress is considered an important factor in atherogenesis. Mammalian cells have a complex network of antioxidants such as catalase, superoxide dismutase, and glutathione peroxidase. However, the mechanisms that regulate the cellular redox state in the vessel wall remain unclear. Recent study has shown that thioredoxin, a thiol-disulfide oxidoreductase, is expressed in atherosclerotic plaques of human carotid arteries. In this study, we investigated the localization and expressional change of glutaredoxin and thioredoxin, two important members of the thiol-disulfide oxidoreductases, in autopsy samples of human coronary arteries. In nonatherosclerotic coronary arteries, glutaredoxin was expressed in endothelial cells, in fibroblasts of the adventitia, and most intensely in medial smooth muscle cells. Interestingly, in atherosclerotic lesions such as hypercellular lesions, the infiltrating macrophages highly expressed glutaredoxin. The expressional pattern of thioredoxin was quite similar to that of glutaredoxin. Western blot analysis demonstrated that hydrogen peroxide stimulated the expression of glutaredoxin in a time- and dose-dependent manner in cultured human coronary artery smooth muscle cells. Fluorescence microtopography with dihydroethidium demonstrated that the generation of reactive oxygen species was associated with the expression of glutaredoxin. These results suggest the possible involvement of thiol-disulfide oxidoreductases in antioxidant protection in human coronary arteries.
Experimental Abdominal Aortic Aneurysms in Mice Lacking Expression of Inducible Nitric Oxide Synthase
Authors: Lee Jason K. , Borhani Martin , Ennis Terri L. , Upchurch Gilbert R. , Thompson Robert W.
Published: September 2007

To determine if nitric oxide synthase (NOS) contributes to the pathophysiology of abdominal aortic aneurysms (AAAs), C57BL/6J mice underwent transient aortic injury to induce a chronic inflammatory response. Wild-type mice developed a significant increase in aortic diameter within 14 days of elastase perfusion (115±16%, 40% incidence of AAAs), along with intense and widespread staining for nitrotyrosine, mononuclear inflammation, and delayed destruction of the elastic lamellae. Expression of both endothelial and neuronal forms of NOS was substantially decreased within AAAs, whereas inducible NOS (iNOS) mRNA was increased 360%, and the enzyme was localized to infiltrating inflammatory cells. By using mice with targeted deletion of iNOS to evaluate the functional importance of this enzyme, male iNOS
−/−
mice developed the same extent of aneurysmal dilatation as congenic controls (121±22%, 40% incidence of AAAs) and exhibited similar structural features except for diminished nitrotyrosine staining. Aneurysmal dilatation was actually enhanced in female iNOS
−/−
mice (141±16%, 80% incidence of AAAs;
P
<0.05), but this effect was reversed by previous oophorectomy. Although extensive protein nitration and increased expression of iNOS accompany the development of elastase-induced experimental AAAs, iNOS is not required in this process and its absence may be deleterious.
Induction of Nuclear Orphan Receptor NGFI-B Gene and Apoptosis in Rat Vascular Smooth Muscle Cells Treated With Pyrrolidinedithiocarbamate
Authors: Watanabe Tokumitsu , Yoshizumi Masao , Akishita Masahiro , Eto Masato , Toba Kenji , Hashimoto Masayoshi , Nagano Koichiro , Liang Yi-Qiang , Ohike Yumiko , Iijima Katsuya , Sudoh Noriko , Kim Seungbum , Nakaoka Takashi , Yamashita Naohide , Ako Junya , Ouchi Yasuyoshi
Published: September 2007

NGFI-B is one of the orphan nuclear receptors, and its gene is implicated in the apoptosis of T cells. The aim of this study was to investigate the expression and the role of NGFI-B in vascular smooth muscle cells (VSMCs). Pyrrolidinedithiocarbamate (PDTC) is a modulator of an oxidative state and is reported to induce apoptosis only when the density of VSMCs is low. Under low VSMC density (10 000 cells/cm
2
), addition of PDTC (0.1 to 10 μmol/L) caused apoptosis of VSMCs, which was confirmed by Hoechst 33258 staining under fluorescence microscopy. At low VSMC density, expression of NGFI-B mRNA was induced 1 hour after the addition of PDTC, peaking at 6 hours, and persisted for up to 12 hours. The protein level of NGFI-B was increased 4 hours after PDTC addition and persisted for up to 12 hours. Under low VSMC density, PDTC-induced expression of NGFI-B mRNA was correlated with the magnitude of apoptosis, which was quantified by enzyme immunoassay for histone-associated DNA fragments. In contrast, when the density of VSMCs was high (50 000 cells/cm
2
), PDTC did not induce apoptosis, and the expression of NGFI-B was only transient. This transient expression pattern was also seen when VSMCs were treated with phorbol ester, calcium ionophore, hydrogen peroxide, or angiotensin II, even at low cell density. We next investigated whether the NGFI-B gene may act as a transcription factor under treatment with PDTC by measuring the promoter activity of luciferase reporter plasmids that contained typical NGFI-B–responsive elements. The PDTC-induced transcriptional activity of NGFI-B was 2-fold higher at low cell density than at high cell density. These data demonstrate that NGFI-B can be induced in VSMCs and suggest that NGFI-B may play a role in PDTC-induced VSMC apoptosis.
Persistent Activation of Nuclear Factor-κB by Interleukin-1β and Subsequent Inducible NO Synthase Expression Requires Extracellular Signal-Regulated Kinase
Authors: Jiang Bingbing , Brecher Peter , Cohen Richard A.
Published: September 2007

Abstract
—

The role of extracellular signal-regulated kinase (ERK) was studied in the signaling pathway by which interleukin-1β (IL-1β) increases the expression of inducible NO synthase (iNOS) in rat vascular smooth muscle cells. IL-1β induced a rapid and transient activation of nuclear factor-κB (NF-κB), followed by a prolonged activation of NF-κB that was required to induce iNOS expression. Either PD98059 or U0126, selective inhibitors of ERK activation, did not influence IL-1β-induced early activation but effectively reduced the prolonged activation of NF-κB and significantly reduced IL-1β induction of iNOS. Transfection with antisense, but not sense, phosphorothioate-modified oligodeoxynucleotides directed toward ERK also reduced IL-1β-induced prolonged NF-κB activation and iNOS expression. IκBβ, but not IκBα degradation, induced by IL-1β was markedly attenuated when ERK activation was inhibited and could be partially responsible for the persistent NF-κB activation. These data suggest that ERK activity is required for persistent NF-κB activation by IL-1β that is necessary for iNOS gene expression.
Increased Rate of Apoptosis in Intimal Arterial Smooth Muscle Cells Through Endogenous Activation of TNF Receptors
Authors: Niemann-Jönsson Audrey , Ares Mikko P.S. , Yan Zhong-Qun , Bu D.-X. , Fredrikson Gunilla Nordin , Brånén Lena , Pörn-Ares Isabella , Nilsson Anna Hultgårdh , Nilsson Jan
Published: September 2007

Abstract
—

Intimal proliferation of smooth muscle cells (SMCs) is a key event in the vascular response to injury, including the early stages of atherosclerosis and restenosis after angioplasty. Tumor necrosis factor-α (TNF-α) has been reported to stimulate growth of cultured human SMCs, but activation of TNF receptors is also known to induce cell death by apoptosis. We report here that SMCs isolated from the neointima of injured rat aortas are characterized by increased expression of TNF-α in response to interleukin-1β and γ-interferon compared with medial SMCs. Basal and serum-stimulated DNA synthesis was higher in intimal than in medial SMCs. In contrast to previous findings on human SMCs, exposure to interleukin-1β/γ-interferon or TNF-α did not affect the growth of rat medial SMCs, inhibited DNA synthesis, and decreased cell numbers in cultures of intimal SMCs. Incubation of intimal SMCs with these cytokines also resulted in induction of terminal dUTP nick end-labeling positivity and caspase-3 expression, suggesting cell death by apoptosis, whereas medial cells were markedly less sensitive in this respect. Cytokine-induced apoptosis in intimal cells was effectively inhibited by treatment with antibodies against TNF receptors. These findings suggest that endogenous activation of TNF receptors may represent a way to limit accumulation of SMCs in injured arteries. This mechanism may also be important in SMC death in advanced atherosclerotic plaques.
Mast Cell Chymase Inhibits Smooth Muscle Cell Growth and Collagen Expression In Vitro
Authors: Wang Yenfeng , Shiota Naotaka , Leskinen Markus J. , Lindstedt Ken A. , Kovanen Petri T.
Published: September 2007

Abstract
—

In the vulnerable areas of fibrous caps of advanced atherosclerotic lesions, chymase-containing mast cells are present. In such areas, the numbers of smooth muscle cells (SMCs) and the content of collagen are reduced. In this in vitro study, we found that the addition of chymase, isolated and purified from rat serosal mast cells, to cultured rat aortic SMCs of the synthetic phenotype (s-SMCs) inhibited their proliferation by blocking the G
0
/G
1
→S transition in the cell cycle. Rat chymase and recombinant human chymase inhibited the expression of collagen type I and type III mRNA in s-SMCs and in human coronary arterial SMCs. The growth-inhibitory effect of chymase was partially reversed by addition to the culture medium of an antibody capable of neutralizing the activity of transforming growth factor-β1 (TGF-β1). Immunocytochemistry showed that the s-SMCs expressed and synthesized extracellular matrix-associated TGF-β1. On exposure to mast cell chymase, the extracellular matrix-associated latent TGF-β1 was released and activated, as demonstrated by immunoblotting and by an ELISA with TGF-β1 type II receptor for capture. When added to s-SMCs, such chymase-released TGF-β1 was capable of inhibiting their growth. In contrast, the inhibitory effect of chymase on collagen synthesis by s-SMCs did not depend on TGF-β1. Taken together, the findings support the hypothesis that chymase released from activated mast cells in atherosclerotic plaques contributes to cap remodeling.
Temporal Expression of Heat Shock Proteins 60 and 70 at Lesion-Prone Sites During Atherogenesis in ApoE-Deficient Mice
Authors: Kanwar Rupinder K. , Kanwar Jagat R. , Wang Dongmao , Ormrod Douglas J. , Krissansen Geoffrey W.
Published: September 2007

In the study, we investigate whether the expressions of heat shock protein (hsp)60 (a potential autoantigen) and the stress-inducible form of cytoprotector hsp70 are correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E–deficient (apoE
−/−
) mice. The apoE
−/−
mouse model is advantageous because the stress-inducible form of hsp70 is not constitutively expressed in mice, unlike primates; hence, tissues under stress can be clearly defined. Both mammalian hsps were detected newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia at lesion-prone sites of 3-week-old apoE
−/−
mice. In 8- and 20-week-old mice, they were strongly and heterogeneously expressed in early to advanced fibrofatty plaques, with levels correlating with lesion severity. Expression was markedly downregulated in advanced collagenous, acellular, calcified plaques of 40- and 69-week-old mice and was absent in control aortas of normocholesterolemic wild-type (apoE
+/+
) mice. Western blot analysis of tissue homogenates confirmed the temporal expression of the hsps. Double immunostaining revealed that both hsps were expressed by lesional endothelial cells, macrophages, smooth muscle cells, and CD3
+
T lymphocytes. This study provides evidence that hsp60 and hsp70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis, suggesting that few cells escape the toxic environment of the atherosclerotic plaque.
Angiotensin I-Converting Enzyme Modulates Neutral Endopeptidase Activity in the Rat
Authors: Oliveri Carla , Ocaranza María Paz , Campos Ximena , Lavandero Sergio , Jalil Jorge E.
Published: September 2007

Abstract
—

— Angiotensin I is a substrate for both ACE and for neutral endopeptidase 24.11 (NEP). We hypothesized that high ACE expression is related to low NEP activity. Accordingly, circulating and tissue NEP and ACE activities were measured by fluorometry in homozygous rats (F
0
and F
2
) for the Lewis microsatellite allele (LL, low ACE) and for the Brown Norway microsatellite allele (BB, high ACE). Plasma, lung, and aortic ACE activities in F
0
and F
2
were higher in BB rats than in LL rats (
P
<0.01), whereas left ventricular ACE activity was similar in both genotypes. In contrast, NEP activity in the LL group was higher in the serum, aorta, and lungs in F
0
and F
2
homozygous (
P
<0.05). Plasma ACE activity was inversely correlated with serum (
r
=−0.6 and −0.598 in F
0
and F
2
, respectively;
P
<0.03) and lung NEP activities (
r
=−0.77 in F
0
and
r
=−0.59 in F
2
,
P
<0.01). Aortic ACE and NEP activities were also correlated (
r
=−0.696 and −0.584 in F
0
and F
2
, respectively;
P
<0.03). In conclusion, genetically determined high ACE expression in rats is inversely related to tissue NEP activity, which could determine lower angiotensin-(1-7) tissue levels.
Antioxidant Effects of Vitamins C and E Are Associated With Altered Activation of Vascular NADPH Oxidase and Superoxide Dismutase in Stroke-Prone SHR
Authors: Chen Xin , Touyz Rhian M. , Park Jeong Bae , Schiffrin Ernesto L.
Published: September 2007

Abstract
—

— Ascorbic acid (vitamin C) and α-tocopherol (vitamin E) have antioxidant properties that could improve redox-sensitive vascular changes associated with hypertension. We determined whether vitamins C and E influence vascular function and structure in hypertension by modulating activity of NADPH oxidase and superoxide dismutase (SOD). Adult stroke-prone spontaneously hypertensive rats (SHRSP) were divided into 3 groups: control (C; n=6), vitamin C-treated (vit C, 1000 mg/day; n=7), and vitamin E-treated (vit E, 1000 IU/day; n=8). All rats were fed 4% NaCl. Blood pressure was measured weekly. After 6 weeks of treatment, the rats were killed, and mesenteric arteries were mounted as pressurized preparations. Vascular O
2
−
generation and NADPH oxidase activity were measured by chemiluminescence. Vascular SOD activity and plasma total antioxidant status (TAS) were determined spectrophotometrically. Blood pressure increased from 212±7 to 265±6 mm Hg in controls. Treatment prevented progression of hypertension (vit C, 222±6 to 234±14 mm Hg; vit E, 220±9 to 227±10 mm Hg). Acetylcholine-induced vasodilation was improved (
P
<0.05), and media-to-lumen ratio was reduced (
P
<0.05) in the treated rats. O
2
−
was lower in vitamin-treated groups compared with controls (vit C, 10±4 nmol · min
−1
· g
−1
dry tissue weight; vit E, 9.6±3.5 nmol · min
−1
· g
−1
dry tissue weight; C, 21±9 nmol · min
−1
· g
−1
dry tissue weight;
P
<0.05). Both vitamin-treated groups showed significant improvement (
P
<0.01) in TAS. These effects were associated with decreased activation of vascular NADPH oxidase (vit C, 46±10; vit E, 50±9; C, 70±16 nmol · min
−1
· g
−1
dry tissue weight,
P
<0.05) and increased activation of SOD (vit C, 12±2; vit E, 8±1; C, 4.6±1 U/mg;
P
<0.05). Our results demonstrate that vitamins C and E reduce oxidative stress, improve vascular function and structure, and prevent progression of hypertension in SHRSP. These effects may be mediated via modulation of enzyme systems that generate free radicals.