bluuebunny/crossref_metadata_2025 huggingface dataset:
Trophic Effect of Norepinephrine on Arterial Intima-Media and Adventitia Is Augmented by Injury and Mediated by Different α1-Adrenoceptor Subtypes
Authors: Zhang Hua , Faber James E.
Published: September 2007

In vivo studies have suggested that norepinephrine (NE) directly contributes to normal vascular wall growth and worsening of hypertrophy, atherosclerosis, and restenosis. However, it is unknown whether these effects are secondary to hemodynamic changes caused by systemic NE or α-adrenoceptor (AR) antagonists. Herein, we determined if NE directly stimulates growth of medial smooth muscle cells (SMCs) and adventitial fibroblasts (AFBs) that we have shown express α1-ARs in similar abundance. The rat aorta was isolated before injury, 4 days after, or 12 days after balloon injury, and maintained under circumferential tension in organ culture for 48 hours with 1 μmol/L NE. Intima-media and adventitia were separated and DNA content, protein synthesis, and protein content measured. In uninjured aorta, NE increased DNA and protein content similarly in adventitia, and increased only protein content in intima-media, suggesting AFB proliferation and SMC hypertrophy. In vessels isolated 4 or 12 days after injury, NE increased all 3 endpoints in both layers by up to 20-fold greater than in uninjured vessels. These effects were dose-dependent and were unaffected by α2- or β-AR blockade (except increased DNA content in adventitia that was also inhibited by α2-AR blockade). Intima-media growth was blocked by KMD3213 (α1A-AR antagonist) and adventitial growth by AH11110A (α1B-AR antagonist), whereas BMY7378 (α1D-AR antagonist) had no effect. NE decreased SMC marker proteins (eg, α-smooth muscle actin and desmin) and augmented the changes induced by injury. These data suggest that prolonged stimulation of α1A- and α1B-ARs induces growth of SMCs and AFBs, respectively, that is significantly augmented by injury.
eNOS Activity Is Reduced in Senescent Human Endothelial Cells
Authors: Matsushita Hidetsugu , Chang Edwin , Glassford Alexander J. , Cooke John P. , Chiu Choy-Pik , Tsao Philip S.
Published: September 2007

Advanced age is associated with endothelial dysfunction and increased risk for atherosclerosis. However, the mechanisms for these observed effects are not clear. To clarify the association between aging and loss of endothelial function, young human aortic endothelial cells (HAECs), senescent HAECs transfected with control vector, and immortalized HAECs containing human telomerase reverse transcriptase (hTERT) were compared for expression of endothelial nitric oxide synthase (eNOS) and production of NO. To investigate a specific function modulated by endothelial NO, adhesion of monocytes under basal conditions as well as after exposure to TNF-α was assessed. A decrease in eNOS mRNA, protein, and activity was observed in endothelial cells at senescence as compared with young HAEC; this effect was blunted in hTERT cells. In all cells, shear stress induced a greater increase in the expression of eNOS protein with the final result being higher levels in hTERT compared with senescent cells. Basal monocyte binding was significantly elevated on aged endothelial cells compared with parental and hTERT cells. Exposure of TNF-α resulted in a 2-fold increase in monocyte adhesion in senescent cells, whereas this effect was reduced in cells transfected with hTERT. Prior exposure to fluid flow significantly reduced subsequent monocyte adhesion in all groups. These studies demonstrate that replicative aging results in decreased endothelial expression of eNOS accompanied by enhanced monocyte binding. Stable expression of hTERT results in endothelial cells with a younger phenotype with greater amount of eNOS and NO activity. Thus, telomerase transfection may have important functional consequences on endothelial cells.
Replacement of the Muscle-Specific Sarcoplasmic Reticulum Ca
2+
-ATPase Isoform SERCA2a by the Nonmuscle SERCA2b Homologue Causes Mild Concentric Hypertrophy and Impairs Contraction-Relaxation of the Heart
Authors: Ver Heyen Mark , Heymans Stephane , Antoons Gudrun , Reed Thomas , Periasamy Muthu , Awede Bonaventure , Lebacq Jean , Vangheluwe Peter , Dewerchin Mieke , Collen Desiré , Sipido Karin , Carmeliet Peter , Wuytack Frank
Published: September 2007

The cardiac sarco(endo)plasmic reticulum Ca
2+
-ATPase gene (
ATP2A2
) encodes the following two different protein isoforms: SERCA2a (muscle-specific) and SERCA2b (ubiquitous). We have investigated whether this isoform specificity is required for normal cardiac function. Gene targeting in mice successfully disrupted the splicing mechanism responsible for generating the SERCA2a isoform. Homozygous
SERCA2a
−/−
mice displayed a complete loss of SERCA2a mRNA and protein resulting in a switch to the SERCA2b isoform. The expression of SERCA2b mRNA and protein in hearts of
SERCA2a
−/−
mice corresponded to only 50% of wild-type SERCA2 levels. Cardiac phospholamban mRNA levels were unaltered in
SERCA2a
−/−
mice, but total phospholamban protein levels increased 2-fold. The transgenic phenotype was characterized by a ≈20% increase in embryonic and neonatal mortality (early phenotype), with histopathologic evidence of major cardiac malformations. Adult
SERCA2a
−/−
animals (adult phenotype) showed a reduced spontaneous nocturnal activity and developed a mild compensatory concentric cardiac hypertrophy with impaired cardiac contractility and relaxation, but preserved β-adrenergic response. Ca
2+
uptake levels in
SERCA2a
−/−
cardiac homogenates were reduced by ≈50%. In isolated cells, relaxation and Ca
2+
removal by the SR were significantly reduced. Comparison of our data with those obtained in mice expressing similar cardiac levels of SERCA2a instead of SERCA2b indicate the importance of the muscle-specific SERCA2a isoform for normal cardiac development and for the cardiac contraction-relaxation cycle.
β
2
-Adrenergic Receptor Overexpression Increases Alveolar Fluid Clearance and Responsiveness to Endogenous Catecholamines in Rats
Authors: Dumasius Vidas , Sznajder Jacob I. , Azzam Zaher S. , Boja John , Mutlu Gökhan M. , Maron Michael B. , Factor Phillip
Published: September 2007

β-Adrenergic agonists accelerate the clearance of alveolar fluid by increasing the expression and activity of epithelial solute transport proteins such as amiloride-sensitive epithelial Na
+
channels (ENaC) and Na,K-ATPases. Here we report that adenoviral-mediated overexpression of a human β
2
-adrenergic receptor (β
2
AR) cDNA increases β
2
AR mRNA, membrane-bound receptor protein expression, and receptor function (procaterol-induced cAMP production) in human lung epithelial cells (A549). Receptor overexpression was associated with increased catecholamine (procaterol)-responsive active Na
+
transport and increased abundance of Na,K-ATPases in the basolateral cell membrane. β
2
AR gene transfer to the alveolar epithelium of normal rats improved membrane-bound β
2
AR expression and function and increased levels of ENaC (α subunit) abundance and Na,K-ATPases activity in apical and basolateral cell membrane fractions isolated from the peripheral lung, respectively. Alveolar fluid clearance (AFC), an index of active Na
+
transport, in β
2
AR overexpressing rats was up to 100% greater than sham-infected controls and rats infected with an adenovirus that expresses no cDNA. The addition of the β
2
AR-specific agonist procaterol to β
2
AR overexpressing lungs did not increase AFC further. AFC in β
2
AR overexpressing lungs from adrenalectomized or propranolol-treated rats revealed clearance rates that were the same or less than normal, untreated, sham-infected controls. These experiments indicate that alveolar β
2
AR overexpression improves β
2
AR function and maximally upregulates β-agonist–responsive active Na
+
transport by improving responsiveness to endogenous catecholamines. These studies suggest that upregulation of β
2
AR function may someday prove useful for the treatment of pulmonary edema.
Cardiac-Specific External Paths for Lidocaine, Defined by Isoform-Specific Residues, Accelerate Recovery From Use-Dependent Block
Authors: Lee Peter J. , Sunami Akihiko , Fozzard Harry A.
Published: September 2007

Local anesthetic antiarrhythmic drugs block voltage-gated Na
+
channels from the cytoplasmic side. In addition, cardiac Na
+
channels can be also blocked by the membrane-impermeant local anesthetic QX via external paths not present in skeletal muscle or brain channels. Introduction of cardiac isoform-specific residues into wild-type skeletal muscle or brain channels creates access paths for external QX block. These paths should affect the characteristics of use-dependent block by influencing drug on- and off-rates. We investigated the effects of these external paths on drug kinetics of lidocaine, a lipophilic drug of clinical relevance, by studying use-dependent block using a two-electrode voltage clamp in
Xenopus
oocytes. Recovery from use-dependent block was slowed when cardiac isoform-specific residues important for external QX access were mutated to skeletal muscle or brain isoform-specific residues. As the fraction of charged lidocaine was decreased by raising external pH, differences in recovery kinetics diminished, indicating that these mutations mostly influenced block by charged lidocaine molecules. Data were fit into a model in which bound drug distributes into charged and neutral forms based on its pK
a
and external pH with separate dissociation paths and recovery-time constants. These isoform-specific mutations altered the recovery-time constants for the charged molecules with smaller effects on those for the neutral molecules. We conclude that the external egress paths created by isoform-specific residues influence the drug kinetics of lidocaine, and these residues define cardiac-specific external paths for local anesthetic drugs.
Ca2+Elevation Evoked by Membrane Depolarization Regulates G Protein Cycle via RGS Proteins in the Heart
Authors: Ishii Masaru , Inanobe Atsushi , Fujita Satoru , Makino Yasunaka , Hosoya Yukio , Kurachi Yoshihisa
Published: September 2007

Regulators of G protein signaling (RGS), which act as GTPase activators, are a family of cytosolic proteins emerging rapidly as an important means of controlling G protein-mediated cell signals. The importance of RGS action has been verified in vitro for various kinds of cell function. Their in situ modes of action in intact cells are, however, poorly understood. Here we show that an increase in intracellular Ca2+evoked by membrane depolarization controls the RGS action on G protein activation of muscarinic K+(KG) channel in the heart. Acetylcholine-induced KGcurrent exhibits a slow time-dependent increase during hyperpolarizing voltage steps, referred to as “relaxation.” This reflects the relief from the decrease in available KGchannel number induced by cell depolarization. This phenomenon is abolished when an increase in intracellular Ca2+is prevented. It is also abolished when a calmodulin inhibitor or a mutant RGS4 is applied that can bind to calmodulin but that does not accelerate GTPase activity. Therefore, an increase in intracellular Ca2+and the resultant formation of Ca2+/calmodulin facilitate GTPase activity of RGS and thus decrease the available channel number on depolarization. These results indicate a novel and probably general pathway that Ca2+-dependent signaling regulates the G protein cycle via RGS proteins.
Coronary Vasodilation and Improvement in Endothelial Dysfunction With Endothelin ET
A
Receptor Blockade
Authors: Halcox Julian P.J. , Nour Khaled R.A. , Zalos Gloria , Quyyumi Arshed A.
Published: September 2007

The endothelium-derived peptide endothelin-1 (ET-1) causes vasoconstriction predominantly via smooth muscle ET
A
receptor activation. We hypothesized that ET
A
receptor inhibition would improve human coronary vascular function. We studied unobstructed coronary arteries of 44 patients with atherosclerosis or its risk factors. Epicardial diameter (
D
) and Doppler flow velocity were measured, and coronary vascular resistance (CVR) was calculated during intracoronary infusions of acetylcholine (ACH) and sodium nitroprusside (SNP), and during cold pressor testing, before and after a 60-minute intracoronary infusion of the ET
A
receptor antagonist BQ-123. BQ-123 dilated the coronary circulation;
D
increased by 5.6±1.0% (
P
<0.0001), and CVR fell by 12±3% (
P
<0.01). The
D
response to ACH, corrected for the SNP response, improved in segments that constricted with ACH at baseline (
P
=0.03), whereas segments that initially dilated with ACH did not change with BQ-123 (
P
=NS). Improvement in
D
and CVR responses to ACH with BQ-123 inversely correlated with baseline ACH responses (
r
=−0.44 [
P
=0.006] and
r
=−0.78 [
P
=0.001], respectively), indicating greater improvement in those with endothelial dysfunction. Similarly, cold pressor testing–mediated epicardial vasoconstriction (−2.0±1.1%) was reversed after BQ-123 (+1.0±0.7%), especially in dysfunctional segments (from −5.6±0.9% to +2.2±0.9%,
P
<0.001). There was no correlation between any risk factor and the response to BQ-123. An arteriovenous difference in ET-1 levels developed after BQ-123, which was consistent with enhanced cardiac clearance of ET-1, probably via ET
B
receptors. Thus, ET-1 acting via the ET
A
receptor contributes to basal human coronary vasoconstrictor tone and endothelial dysfunction. This suggests that ET
A
receptor antagonism may have therapeutic potential in the treatment of endothelial dysfunction and atherosclerosis.
Smooth Muscle Cell Phenotypic Transition Associated With Calcification
Authors: Steitz Susie A. , Speer Mei Y. , Curinga Gabrielle , Yang Hsueh-Ying , Haynes Paul , Aebersold Ruedi , Schinke Thorsten , Karsenty Gerard , Giachelli Cecilia M.
Published: September 2007

Bovine aortic smooth muscle cell (BASMC) cultures undergo mineralization on addition of the organic phosphate donor, β-glycerophosphate (βGP). Mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles, as has been described in calcified vascular lesions in vivo as well as in bone and teeth. In the present study, we used this model to investigate the molecular mechanisms driving vascular calcification. We found that BASMCs lost their lineage markers, SM22α and smooth muscle α-actin, within 10 days of being placed under calcifying conditions. Conversely, the cells gained an osteogenic phenotype as indicated by an increase in expression and DNA-binding activity of the transcription factor, core binding factor α1 (Cbfa1). Moreover, genes containing the Cbfa1 binding site, OSE2, including osteopontin, osteocalcin, and alkaline phosphatase were elevated. The relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null (MGP
−/−
) mice whose arteries spontaneously calcify. We found that arterial calcification was associated with a similar loss in smooth muscle markers and a gain of osteopontin and Cbfa1 expression. These data demonstrate a novel association of vascular calcification with smooth muscle cell phenotypic transition, in which several osteogenic proteins including osteopontin, osteocalcin, and the bone determining factor Cbfa1 are gained. The findings suggest a positive role for SMCs in promoting vascular calcification.
In Vitro Evidence for an Intracellular Site of Angiotensin Action
Authors: Cook Julia L. , Zhang Zhuo , Re Richard N.
Published: September 2007

To differentiate the relative effects of nuclear and cell surface angiotensin II (Ang II) receptors, we mutated the angiotensinogen cDNA by removing the signal sequence-encoding region to produce a nonsecreted form of angiotensinogen [Ang(−S)Exp]. Rat hepatoma cells (which produce renin and angiotensin-converting enzyme mRNAs) were stably transfected with Ang(−S)Exp/pSVL (or a corresponding control) expression plasmid, and mitotic indices were measured for stably transfected cell lines. Experimental clonal cell lines demonstrate an average of 33±4.4% (
P
<0.001) increase in percentage-labeled nuclei compared with control cell lines. The mitogenic effect is blocked by 10
−6
mol/L losartan and by 1 μmol/L renin antisense phosphorothioate oligomers but not by 10
−6
mol/L candesartan. In addition, phenylarsine oxide, which blocks angiotensin receptor internalization, abolishes the losartan inhibitory effect, suggesting that after cell-surface receptor-mediated endocytosis, losartan blocks Ang II nuclear receptors. PDGF mRNA levels are elevated 2.2-fold in Ang(−S)Exp transfected cell lines; addition of anti-PDGF antibodies to the culture medium partially blocks the mitogenic effect of Ang(−S)Exp, while anti–Ang II antibodies have no effect. These results suggest that the Ang(−S)Exp growth effect is due, in part, to autocrine/paracrine stimulation by secreted PDGF after Ang II/Ang II receptor intracellular interactions. We further demonstrate that these cells produce the alternative renin transcript, renin 1A, which apparently lacks a signal sequence and is maintained intracellularly. Collectively, these studies of cultured cells suggest that some cell types may possess components of the renin-angiotensin system that permit intracellular processing of angiotensinogen to Ang II and that Ang II generated intracellularly may be mitogenic.
Injury Induces Dedifferentiation of Smooth Muscle Cells and Increased Matrix-Degrading Metalloproteinase Activity in Human Saphenous Vein
Authors: Johnson Jason L. , van Eys Guillaume J. J. M. , Angelini Gianni D. , George Sarah J.
Published: September 2007

Abstract
—Long-term patency of human saphenous vein bypass grafts is low because of intimal thickening and superimposed atherosclerosis. Matrix-degrading metalloproteinases (MMPs) and changes in vascular smooth muscle cell (VSMC) phenotype are thought to be essential for the VSMC migration that contributes to intimal thickening. We examined VSMC phenotype and MMP activity in saphenous veins obtained before and after surgical manipulation. Surgical preparation of the veins significantly increased pro-MMP-1 expression by 2-fold and significantly reduced tissue inhibitor of MMPs (TIMP)-2 expression, whereas MMP-3 and TIMP-1 were unaffected. Furthermore, caseinolytic and gelatinolytic activities measured by in situ zymography were dramatically elevated by injury. The expression of desmin and smoothelin was significantly decreased by injury, whereas vimentin expression was significantly increased. In addition, these changes in phenotype and MMP activity were localized to a subpopulation of VSMCs, the circumferential medial VSMCs. Our data show that surgical preparative injury induces phenotypic modulation of a subpopulation of medial VSMCs to a synthetic phenotype and increases MMP activity. This may favor matrix degradation, VSMC migration, and the subsequent intimal thickening that leads to graft failure.
Blood Flow Decrease Induces Apoptosis of Endothelial Cells in Previously Dilated Arteries Resulting From Chronic High Blood Flow
Authors: Sho Eiketsu , Sho Mien , Singh Tej M. , Xu Chengpei , Zarins Christopher K. , Masuda Hirotake
Published: September 2007

Abstract
—We investigated apoptosis of endothelial cells during the arterial narrowing process in response to reduction in flow. The decrease in flow was created in the carotid artery by closure of an arteriovenous fistula (AVF), which had been established for 28 days in rabbits. The endothelial cell apoptosis in the carotid artery was studied at 1, 3, 7, and 21 days of flow reduction after closure of the AVF by use of terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) with laser scanning confocal microscopy and transmission and scanning electron microscopy. After AVF closure, arterial lumen diameter was reduced by 36%, and compared with endothelial cells before the closure, the number of endothelial cells was decreased by 45% at 21 days. Endothelial cell apoptosis was observed at 1 day, peaked at 3 days (381.3±87.1 cells per square millimeter), and decreased at 7 days. These cells had irregular protrusions under scanning electron microscopy and were characterized by fragmented nuclei under transmission electron microscopy. Apoptotic cells were mainly beneath the endothelium and were occasionally within smooth muscle cells and endothelial cells. The results suggest that apoptosis of endothelial cells may play a role in the arterial remodeling in response to a reduction in flow.
Tranilast Inhibits Cardiac Allograft Vasculopathy in Association With p21
Waf1/Cip1
Expression on Neointimal Cells in Murine Cardiac Transplantation Model
Authors: Izawa Atsushi , Suzuki Jun-ichi , Takahashi Wataru , Amano Jun , Isobe Mitsuaki
Published: September 2007

Abstract
—Cardiac allograft vasculopathy is a major complication after cardiac transplantation, often limiting long-term recipient survival.
N
-(3,4-Dimethoxycinnamoyl)anthranilic acid (tranilast) inhibits cyclin-dependent kinase activity through p21
Waf1/Cip1
induction and arrests vascular smooth muscle cell proliferation in vitro. We tested a hypothesis that tranilast inhibits the vasculopathy characterized by diffuse intimal thickening in a murine heart transplantation model. Hearts from DBA/2 mice were heterotopically transplanted into B10.D2 mice as allografts. Oral administration of tranilast started 3 days before transplantation at doses of 550 or 1040 mg/kg per day until the animals were killed. Cardiac allograft vasculopathy was defined as luminal stenosis caused by neointimal formation. The percentage of luminal stenosis and cardiac rejection were analyzed 14 and 28 days after transplantation. Tranilast administration was associated with a marked reduction in luminal occlusion but with no significant effect on cardiac rejection. Immunohistochemical study of the tranilast-treated graft coronary arteries revealed enhancement of p21
Waf1/Cip1
and decreased expression of proliferating cell nuclear antigen in the neointima. The significant reduction in allograft vasculopathy concomitant with the enhancement of p21
Waf1/Cip1
indicates that tranilast has an antiproliferative effect that could be applicable to clinical treatment of cardiac allograft vasculopathy.
Effects of Endurance Exercise Training on Plasma HDL Cholesterol Levels Depend on Levels of Triglycerides
Authors: Couillard Charles , Després Jean-Pierre , Lamarche Benoît , Bergeron Jean , Gagnon Jacques , Leon Arthur S. , Rao D. C. , Skinner James S. , Wilmore Jack H. , Bouchard Claude
Published: September 2007

Abstract
—High density lipoprotein (HDL) cholesterol concentrations have been shown to increase with regular endurance exercise and, therefore, can contribute to a lower risk of coronary heart disease in physically active individuals compared with sedentary subjects. Although low HDL cholesterol levels are frequently observed in combination with hypertriglyceridemia, some individuals may be characterized by isolated hypoalphalipoproteinemia, ie, low HDL cholesterol levels in the absence of elevated triglyceride (TG) concentrations. The present study compared the responses of numerous lipoprotein-lipid variables to a 20-week endurance exercise training program in men categorized on the basis of baseline TG and HDL cholesterol concentrations: (1) low TG and high HDL cholesterol (normolipidemia), (2) low TG and low HDL cholesterol (isolated low HDL cholesterol), (3) high TG and high HDL cholesterol (isolated high TGs), and (4) high TGs and low HDL cholesterol (high TG/low HDL cholesterol). A series of physical and metabolic variables was measured before and after the training program in a sample of 200 men enrolled in the Health, Risk Factors, Exercise Training and Genetics (HERITAGE) Family Study. At baseline, men with high TG/low HDL cholesterol had more visceral adipose tissue than did men with isolated low HDL cholesterol and men with normolipidemia. The 0.4% (not significant) exercise-induced increase in HDL cholesterol levels in men with isolated low HDL cholesterol suggests that they did not benefit from the “HDL-raising” effect of exercise. In contrast, men with high TG/low HDL cholesterol showed a significant increase in HDL cholesterol levels (4.9%,
P
<0.005). Whereas both subgroups of men with elevated TG levels showed reductions in plasma TGs (≈−15.0%,
P
<0.005), only those with high TG/low HDL cholesterol showed significantly reduced apolipoprotein B levels at the end of the study (−6.0%,
P
<0.005). Multiple regression analyses revealed that the exercise-induced change in abdominal subcutaneous adipose tissue (10.6%,
P
<0.01) was the only significant correlate of the increase in plasma HDL cholesterol with training in men with high TG/low HDL cholesterol. Results of the present study suggest that regular endurance exercise training may be particularly helpful in men with low HDL cholesterol, elevated TGs, and abdominal obesity.
Genetic Contributions to Plasma Total Antioxidant Activity
Authors: Wang Xing Li , Rainwater David L. , VandeBerg Jane F. , Mitchell Braxton D. , Mahaney Michael C.
Published: September 2007

Abstract
—Oxidative stress plays important roles in a wide spectrum of pathological processes, such as atherosclerosis. Although several environmental factors are documented to influence redox metabolism, relatively little is known about genetic effects. In the present study, we evaluated genetic contributions to variation in plasma total antioxidant status (TAS), a measure of peroxyl-scavenging capacity, in 1337 members of 40 Mexican American families. TAS levels were significantly lower in women than in men (1.675±0.004 versus 1.805±0.005 mmol/L, respectively;
P
<0.001), and there was a significant decline of TAS levels with age in men but not in women (
P
<0.01 for the interaction). Quantitative genetic analysis indicated the heritability of TAS levels to be 0.509±0.052; ie, ≈51% of the residual variance (after covariate adjustment) in TAS levels was due to the additive effects of genes (
P
<0.001). We have further observed a significant gene-by-smoking interaction (
P
<0.05). Additive genetic effects account for 83% of the residual phenotypic variance in TAS levels among smokers, but they account for only 49% in nonsmokers. However, genes contributing to TAS variation are the same in smokers and nonsmokers. Our study for the first time demonstrates that TAS, an indicator of redox homeostasis, is under strong genetic control, especially among smokers. With appropriate tools, such as genome screening, it should be possible to localize genes that regulate redox homeostasis and, ultimately, identify the DNA sequence variants predisposing subjects to oxidative damage.
Replacement of Dietary Saturated Fatty Acids by
Trans
Fatty Acids Lowers Serum HDL Cholesterol and Impairs Endothelial Function in Healthy Men and Women
Authors: de Roos Nicole M. , Bots Michiel L. , Katan Martijn B.
Published: September 2007

Abstract
—We tested whether
trans
fatty acids and saturated fatty acids had different effects on flow-mediated vasodilation (FMD), a risk marker of coronary heart disease (CHD). Consumption of
trans
fatty acids is related to increased risk of CHD, probably through effects on lipoproteins.
Trans
fatty acids differ from most saturated fatty acids because they decrease serum high-density lipoprotein (HDL) cholesterol, and this may increase the risk of CHD. We fed 29 volunteers 2 controlled diets in a 2×4-week randomized crossover design. The “Trans-diet” contained 9.2 energy percent of
trans
fatty acids; these were replaced by saturated fatty acids in the “Sat-diet.” Mean serum HDL cholesterol after the Trans-diet was 0.39 mmol/L (14.8 mg/dL), or 21% lower than after the Sat-diet (95% CI 0.28 to 0.50 mmol/L). Serum low density lipoprotein and triglyceride concentrations were stable. FMD+SD was 4.4±2.3% after the Trans-diet and 6.2±3.0% after the Sat-diet (difference −1.8%, 95% CI −3.2 to −0.4). Replacement of dietary saturated fatty acids by
trans
fatty acids impaired FMD of the brachial artery, which suggests increased risk of CHD. Further studies are needed to test whether the decrease in serum HDL cholesterol caused the impairment of FMD.
Interleukin-15 Expression in Atherosclerotic Plaques
Authors: Houtkamp Mischa A. , van der Wal Allard C. , de Boer Onno J. , van der Loos Chris M. , de Boer Piet A. J. , Moorman Antoon F. M. , Becker Anton E.
Published: September 2007

Abstract
—T-cell activation in atherosclerotic plaques is thought to be initiated by plaque-derived antigens, such as oxidized LDL (oxLDL). An alternative pathway of T-cell activation independent of antigen stimulation, mediated by the cytokine interleukin (IL)-15, was recently described. We investigated IL-15 expression in atherosclerotic plaques in relation to plaque morphology, inflammatory cells, T-cell activation, and oxidation-specific epitopes by use of immunohistochemistry. In situ hybridization was used to evaluate IL-15 mRNA expression. We also studied the proliferative response of plaque-derived T-cell lines to IL-15 in vitro using [
3
H]thymidine incorporation. Fresh-frozen specimens were classified as fibrous (n=9), fibrolipid (n=8), and lipid-rich (n=14) plaques; normal vessels (n=4) served as reference. Expression of IL-15 mRNA and protein was found almost solely in fibrolipid and lipid-rich plaques, associated with oxLDL-positive macrophages. Sequential immunostains revealed colocalization between IL-15– and CD40L-positive T cells. Moreover, plaque-derived T-cell lines were highly responsive to IL-15. Hence, IL-15 could provide a pathway for antigen-independent T-cell activation.
NO-Dependent Vasorelaxation Is Impaired After Gene Transfer of Inducible NO-Synthase
Authors: Gunnett Carol A. , Lund Donald D. , Chu Yi , Brooks Robert M. , Faraci Frank M. , Heistad Donald D.
Published: September 2007

Proinflammatory stimuli produce expression of inducible NO-synthase (iNOS) within blood vessels and are associated with impaired endothelium-dependent relaxation. Gene transfer of iNOS was used to test the hypothesis that expression of iNOS in blood vessels produces impairment of NO-dependent relaxation as well as contraction. An adenoviral vector containing cDNA for murine iNOS, AdCMViNOS, and a control virus, AdCMVBglII, were used for gene transfer to rabbit carotid arteries in vitro and in vivo. After gene transfer of iNOS in vitro, contractile responses to KCl, phenylephrine, and U46619 were impaired. Relaxation in response to acetylcholine, ADP, A23187, and nitroprusside was also impaired. For example, maximum relaxation of vessels to acetylcholine (10 μmol/L) was 78±4% (mean±SE) after AdBglII (10
10.5
plaque-forming units) and 34±5% after AdiNOS (10
10.5
plaque-forming units,
P
<0.05). NO-independent relaxation in response to 8-bromo-cGMP and papaverine was not impaired after AdiNOS. Contraction and relaxation were improved in carotid arteries expressing iNOS by aminoguanidine and
l
-
N
-iminoethyl lysine, inhibitors of iNOS. After intraluminal gene transfer of iNOS in vivo, contraction of vessels in vitro was normal, but responses to acetylcholine were impaired. In summary, the major finding is that NO-dependent relaxation is impaired in arteries after gene transfer of iNOS in vitro and in vivo. Thus, expression of iNOS per se impairs NO-dependent relaxation.
Compensatory Enlargement and Stenosis Develop in ApoE
−/−
and ApoE*3-Leiden Transgenic Mice
Authors: Lutgens Esther , de Muinck Ebo D. , Heeneman Sylvia , Daemen Mat J.A.P.
Published: September 2007

Atherosclerotic mouse models develop little ischemic organ damage and no infarctions, despite the presence of large atherosclerotic lesions. Therefore, we hypothesize that luminal changes do not follow atherosclerotic lesion development. Because a phenomenon that may explain the discrepancy between luminal changes and lesion size is vascular remodeling, we measured parameters of vascular remodeling in the carotid arteries (CAs), thoracic aorta (TA), and abdominal aorta (AA) of apolipoprotein E (apoE)–deficient (apoE
−/−
) and apoE*3-Leiden mice, 2 well-known mouse models of atherosclerosis. Atherosclerotic lesions were classified (American Heart Association [AHA] types II through V), and plaque thickness, compensatory enlargement versus constrictive remodeling, lumen diameter, stenosis, and media thickness were measured relative to the nondiseased arterial wall. In CAs, plaque thickness increased during atherogenesis. CAs showed compensatory enlargement (apoE
−/−
55%, apoE*3-Leiden 38%). Regression analysis revealed a positive correlation between plaque and lumen area (for apoE
−/−
,
R
=0.95; for apoE*3-Leiden,
R
=0.90). Medial thinning and elastolysis were also observed. During atherogenesis, lumen diameter decreased (apoE
−/−
−69%, apoE*3-Leiden −40%), and stenosis >70% developed. TA and AA showed similar features, but neither developed a progressive decrease in lumen diameter or stenosis >70%. In CAs, TA, and AA of apoE
−/−
and apoE*3-Leiden mice, atherogenesis is associated with compensatory enlargement, medial thinning, and elastolysis. A progressive decrease in lumen diameter and stenoses >70% occur only in CAs. Vascular remodeling is more prominent in apoE
−/−
mice.
Antioxidant Supplements Block the Response of HDL to Simvastatin-Niacin Therapy in Patients With Coronary Artery Disease and Low HDL
Authors: Cheung Marian C. , Zhao Xue-Qiao , Chait Alan , Albers John J. , Brown B. Greg
Published: September 2007

One strategy for treating coronary artery disease (CAD) patients with low HDL cholesterol (HDL-C) is to maximally increase the HDL-C to LDL-C ratio by combining lifestyle changes with niacin (N) plus a statin. Because HDL can prevent LDL oxidation, the low-HDL state also may benefit clinically from supplemental antioxidants. Lipoprotein changes over 12 months were studied in 153 CAD subjects with low HDL-C randomized to take simvastatin and niacin (S-N), antioxidants (vitamins E and C, β-carotene, and selenium), S-N plus antioxidants (S-N+A), or placebo. Mean baseline plasma cholesterol, triglyceride, LDL-C, and HDL-C levels of the 153 subjects were 196, 207, 127, and 32 mg/dL, respectively. Without S-N, lipid changes were minor. The S-N and S-N+A groups had comparably significant reductions (
P
≤0.001) in plasma cholesterol, triglyceride, and LDL-C. However, increases in HDL-C, especially HDL
2
-C, were consistently higher in the S-N group than in the S-N+A group (25% vs 18% and 42% vs 0%, respectively). With S-N, but not with S-N+A, there was a selective increase in apolipoprotein (apo) A-I (64%) in HDL particles containing apo A-I but not A-II [Lp(A-I)] and their particle size. Thus, in CAD patients with low HDL-C, S-N substantially increased HDL
2
-C, Lp(A-I), and HDL particle size. These favorable responses were blunted by the antioxidants used owing to a striking selective effect on Lp(A-I). This unexpected adverse interaction between antioxidants and lipid therapy may have important implications for the management of CAD.
In Vivo Magnetic Resonance Imaging of Experimental Thrombosis in a Rabbit Model
Authors: Johnstone Michael T. , Botnar René M. , Perez Alexandra S. , Stewart Robert , Quist William C. , Hamilton James A. , Manning Warren J.
Published: September 2007

The process of atherosclerotic plaque disruption has been difficult to monitor because of the lack of an animal model and the limited ability to directly visualize the plaque and overlying thrombus in vivo. Our aim was to validate in vivo magnetic resonance imaging (MRI) of the thrombus formation after pharmacological triggering of plaque disruption in the modified Constantinides animal model of plaque disruption. Atherosclerosis was induced in 9 New Zealand White male rabbits (3 kg) with aortic balloon endothelial injury followed by a high cholesterol (1%) diet for 8 weeks. After baseline (pretrigger) MRI, the rabbits underwent pharmacological triggering with Russell’s viper venom and histamine, followed by another MRI 48 hours later. Contiguous cross-sectional T2-weighted fast spin echo images of the abdominal aorta were compared by histopathology. In all animals, aortic wall thickening was present on the pretrigger MRI. On MRIs performed 48 hours after triggering, a histologically confirmed intraluminal thrombus was visualized in 6 (67%) of the 9 animals. MRI data correlated with the histopathology regarding aortic wall thickness (
R
=0.77,
P
<0.0005), thrombus size (
R
=0.82,
P
<0.0001), thrombus length (
R
=0.86,
P
<0.005), and anatomic location (
R
=0.98,
P
<0.0001). In vivo, MRI reliably determines the presence, location, and size of the thrombus in this animal model of atherosclerosis and plaque disruption. The combination of in vivo MRI and the modified Constantinides animal model could be an important research tool for our understanding of the pathogenesis of acute coronary syndromes.
Paraoxonase Status in Coronary Heart Disease
Authors: Mackness Bharti , Davies Gershan K. , Turkie Wajdi , Lee Evelyn , Roberts David H. , Hill Elizabeth , Roberts Chris , Durrington Paul N. , Mackness Michael I.
Published: September 2007

Human serum paraoxonase (PON1) hydrolyzes oxidized lipids in low density lipoprotein (LDL) and could therefore retard the development of atherosclerosis. In keeping with this hypothesis, several case-control studies have shown a relationship between the presence of coronary heart disease (CHD) and polymorphisms at amino acid positions 55 and 192 of PON1, which we associated with a decreased capacity of PON1 to protect LDL against the accumulation of lipid peroxides, but some other studies have not. However, the PON1 polymorphisms are only 1 factor in determining the activity and concentration of the enzyme. Only 3 of the previous 18 studies directly determined PON1 activity and concentration. Therefore, we studied PON1 activity, concentration, and gene distribution in 417 subjects with angiographically proven CHD and in 282 control subjects. We found that PON1 activity and concentration were significantly lower in subjects with CHD than in control subjects (activity to paraoxon 122.8 [3.3 to 802.8] versus 214.6 [26.3 to 620.8] nmol · min
−1
· mL
−1
,
P
<0.001; concentration 71.6 [11.4 to 489.3] versus 89.1 [16.8 to 527.4] μg/mL,
P
<0.001). There were no differences in the PON1-55 and -192 polymorphisms or clusterin concentration between patients with CHD and control subjects. These results indicate that lower PON1 activity and concentration and, therefore, the reduced ability to prevent LDL lipid peroxidation may be more important in determining the presence of CHD than paraoxonase genetic polymorphisms.
Characterization of a Mouse Model for Thrombomodulin Deficiency
Authors: Weiler Hartmut , Lindner Volkhard , Kerlin Bryce , Isermann Berend H. , Hendrickson Sara B. , Cooley Brian C. , Meh David A. , Mosesson Michael W. , Shworak Nicholas W. , Post Mark J. , Conway Edward M. , Ulfman Laurien H. , von Andrian Ulrich H. , Weitz Jeffrey I.
Published: September 2007

Mutations in the gene encoding thrombomodulin (TM), a thrombin regulator, are suspected risk factors for venous and arterial thrombotic disease. We have previously described the generation of TM
Pro/Pro
mice carrying a TM gene mutation that disrupts the TM-dependent activation of protein C. Here, it is shown that inbred C57BL/6J TM
Pro/Pro
mice exhibit a hypercoagulable state and an increased susceptibility to thrombosis and sepsis. Platelet thrombus growth after FeCl
3
-induced acute endothelial injury was accelerated in mutant mice. Vascular stasis after permanent ligation of the carotid artery precipitated thrombosis in mutant but not in normal mice. Mutant mice showed increased mortality after exposure to high doses of endotoxin and demonstrated altered cytokine production in response to low-dose endotoxin. The severity of the hypercoagulable state and chronic microvascular thrombosis caused by the TM
Pro
mutation is profoundly influenced by mouse strain-specific genetic differences between C57BL/6 and 129SvPas mice. These data demonstrate that in mice, TM is a physiologically relevant regulator of platelet- and coagulation-driven large-vessel thrombosis and modifies the response to endotoxin-induced inflammation. The phenotypic penetrance of the TM
Pro
mutation is determined by as-yet-uncharacterized genetic modifiers of thrombosis other than TM.
Exercise Prevents the Accumulation of Triglyceride-Rich Lipoproteins and Their Remnants Seen When Changing to a High-Carbohydrate Diet
Authors: Koutsari Christina , Karpe Fredrik , Humphreys Sandy M. , Frayn Keith N. , Hardman Adrianne E.
Published: September 2007

We tested the hypothesis that daily aerobic exercise opposes the fasting hypertriglyceridemia and exaggerated postprandial lipemia observed after substituting dietary fat with carbohydrate. Eight healthy postmenopausal women aged 51 to 66 years consumed the same high-fat mixed meal on 3 occasions: (1) after 3 days on a low-carbohydrate diet (35%, 50%, and 15% energy from carbohydrate, fat, and protein, respectively); (2) after 3 days on an isoenergetic high-carbohydrate diet (corresponding values 70%, 15%, and 15%); and (3) after 3 days on the same high-carbohydrate diet with 60 minutes of brisk walking daily. Plasma triglycerides were higher after the high-carbohydrate diet than after the low-carbohydrate diet: fasting, 1.58±0.19 versus 0.96±0.12 mmol/L, respectively; 6-hour postprandial area under concentration versus time curve, 13.74±1.57 versus 10.12±1.15 (mmol/L)×hour, respectively (both
P
<0.01). In the fasted and postprandial states, concentrations of apolipoproteins B-48 and B-100 in the triglyceride-rich lipoprotein fraction were significantly higher after the high-carbohydrate diet, as was the concentration of remnant-like lipoprotein particle cholesterol (a measure of lipoprotein remnants). These carbohydrate-induced increases in the number of circulating triglyceride-rich particles and their remnants were abolished when subjects had exercised daily during the high-carbohydrate diet.
Dietary Supplementation With Methionine and Homocysteine Promotes Early Atherosclerosis but Not Plaque Rupture in ApoE-Deficient Mice
Authors: Zhou Ji , Møller Jan , Danielsen Carl C. , Bentzon Jacob , Ravn Hanne B. , Austin Richard C. , Falk Erling
Published: September 2007

Hyperhomocysteinemia is an independent risk factor for atherothrombosis. However, causality is unproven, and it remains unknown whether hyperhomocysteinemia promotes atherosclerosis, plaque rupture, and/or thrombosis. We evaluated the short- and long-term effects of hyperhomocysteinemia on plaque size and structure in 99 atherosclerosis-prone apolipoprotein E-deficient mice. Hyperhomocysteinemia was induced by methionine (Met) or homocysteine (HcyH) supplementation: low Met (+11 g Met/kg food), high Met (+33 g Met/kg food), low HcyH (0.9 g HcyH/L drinking water), and high HcyH (1.8 g HcyH/L drinking water). Met and HcyH supplementation significantly raised plasma total homocysteine levels by 4- to 16-fold above those observed in mice fed a control diet (up to 146.1 μmol/L). Compared with controls, aortic root plaque size was significantly larger in supplemented groups after 3 months (56% and 173% larger in high-Met and high-HcyH, respectively) but not after 12 months. Hyperhomocysteinemia was associated with an increase in the amount of collagen in plaques after both 3 and 12 months. Mechanical testing of the tail tendons revealed no weakening of collagen after 12 months of hyperhomocysteinemia. Many plaques in both control and supplemented mice appeared rupture prone morphologically, but all aortic root plaques and all but 1 coronary plaque had an intact surface without rupture or thrombosis. Thus, diet-induced hyperhomocysteinemia promotes early atherosclerosis and plaque fibrosis but does not, even in the long term, weaken collagen or induce plaque rupture.
l
-Arginine Administration Reduces Neointima Formation After Stent Injury in Rats by a Nitric Oxide-Mediated Mechanism
Authors: Vermeersch P. , Nong Z. , Stabile E. , Varenne O. , Gillijns H. , Pellens M. , Van Pelt N. , Hoylaerts M. , De Scheerder I. , Collen D. , Janssens S.
Published: September 2007

The clinical outcome of vascular stenting is limited by in-stent stenosis. Increased nitric oxide (NO)/cGMP signaling by
l
-arginine (
l
-Arg) supplementation, the substrate for NO synthase (NOS), or
NOS
gene transfer may reduce in-stent neointima formation. After stenting, vascular cell proliferation in rat carotid arteries, as measured by 5′-bromodeoxyuridine (5′-BrdU) incorporation, indicated 15±8%, 28±5%, and 33±7% 5′-BrdU-positive vascular cells at 4, 7, and 14 days, respectively. Reporter β-galactosidase gene transfer efficacy was evidenced by 30% β-galactosidase-expressing medial smooth muscle cells at 14 days. The intima-to-media ratio (I/M) progressively increased to 2.32±0.24 at 14 days. To target in-stent neointima formation, animals were infected with adenoviral vectors (4×10
10
plaque-forming units per mL) expressing NOS2 (AdNOS2) or no transgene (AdRR5), or they received daily doses of
l
-Arg (500 mg · kg
−1
·
d−1
IP). The neointima at 14 days was smaller in
l
-Arg-treated than in untreated rats (I/M 1.25±0.35 vs 2.32±0.24,
P
<0.05, n=7 each) or in AdRR5- and AdNOS2-infected rats (I/M 2.57±0.43, n=7 and 1.82±0.75, n=8, respectively;
P
<0.05 for both). The effect of
l
-Arg was abolished by simultaneous administration of
N
G
-nitro
l
-arginine methyl ester, an NOS inhibitor (2.03±0.39,
P
<0.05, vs
l
-Arg). Inflammation was markedly less in
l
-Arg- and AdNOS2-treated than in AdRR5-infected rats. Supplemental
l
-Arg reduces neointima formation after stenting by way of an NOS-dependent mechanism and may be a valuable strategy to target in-stent stenosis.
Expression of SR-PSOX, a Novel Cell-Surface Scavenger Receptor for Phosphatidylserine and Oxidized LDL in Human Atherosclerotic Lesions
Authors: Minami Manabu , Kume Noriaki , Shimaoka Takeshi , Kataoka Hiroharu , Hayashida Kazutaka , Akiyama Yoshinori , Nagata Izumi , Ando Kenji , Nobuyoshi Masakiyo , Hanyuu Michiya , Komeda Masashi , Yonehara Shin , Kita Toru
Published: September 2007

Receptor-mediated endocytosis of oxidized low density lipoprotein (Ox-LDL) by macrophages and the subsequent foam cell transformation in the arterial intima are key events in early atherogenesis. Recently, we have identified a novel macrophage cell-surface receptor for Ox-LDL by expression cloning from a cDNA library of phorbol 12-myristate 13-acetate–stimulated THP-1 cells, designated as the scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX). Here, we examined SR-PSOX expression in human atherosclerotic lesions. Total cellular RNA and fresh frozen sections were prepared from human carotid endarterectomy specimens (from 21 patients) and directional coronary atherectomy specimens (from 11 patients). Fragments of human aortas of 2 patients without visible atherosclerotic lesions served as negative controls. Quantitative reverse transcription–polymerase chain reaction demonstrated that SR-PSOX mRNA expression was prominent in atherosclerotic lesions but undetectable in normal aortas. Immunohistochemistry showed that SR-PSOX was predominantly expressed by lipid-laden macrophages in the intima of atherosclerotic plaques in carotid endarterectomy and directional coronary atherectomy specimens, although its expression was not detectable in normal arterial wall. Double-labeled immunohistochemistry confirmed that SR-PSOX is expressed by intimal macrophages. Taken together, SR-PSOX may be involved in Ox-LDL uptake and subsequent foam cell transformation in macrophages in vivo and thus may play important roles in human atherosclerotic lesion formation.