bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation by Fibrinogen and Its Products of Intercellular Adhesion Molecule-1 Expression in Human Saphenous Vein Endothelial Cells
Authors: Harley Suzanne L. , Sturge Justin , Powell Janet T.
Published: June 2011

Abstract
—It has been reported that fibrinogen may act as a bridging ligand, binding to intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells and to Mac-1 on THP-1 cells (a monocytic cell line) to increase adhesion. In this study, we investigated whether fibrinogen altered the expression of ICAM-1 and, thus, increased the adhesion of THP-1 cells to cultured human saphenous vein endothelial cells (HSVECs). Incubation of HSVECs with 0.3 to 4 μmol/L fibrinogen caused a time- and concentration-dependent increase in ICAM-1, as determined by ELISA. The 4- to 5-fold increase in ICAM-1 protein concentration in HSVECs stimulated by 4 μmol/L fibrinogen for 6 hours was concomitant with a 4- to 5-fold increase in ICAM-1 mRNA. This fibrinogen-stimulated ICAM-1 upregulation was associated with a 2-fold increase in THP-1 cell adhesion to HSVECs. The fibrinogen-derived peptide Bβ15-42 bound to HSVECs (
K
d
0.18 μmol/L). Preincubation of HSVECs with Bβ15-42, a neutralizing antibody to urokinase plasminogen activator (uPA), or the F(ab)
1
fragment of a monoclonal antibody to vascular endothelial cadherin significantly attenuated the increase in ICAM-1 stimulated by fibrinogen. Capillary electrophoretic analysis indicated that anti-uPA prevented the release of any fibrinopeptide B (Bβ1-14) in cultures of HSVECs incubated with 4 μmol/L fibrinogen for 6 hours. Moreover, incubation of HSVECs with either fibrin monomer (1 μmol/L) or monoclonal antibodies to vascular endothelial cadherin (25 μg/mL) increased ICAM-1 protein concentration 3- to 4-fold. These findings indicate that cleavage of fibrinopeptide B from fibrinogen by endothelial uPA permits the exposed Bβ15-42 sequence of fibrinogen to bind to vascular endothelial cadherin on HSVECs and to upregulate the expression of ICAM-1.
Oxidized Low Density Lipoprotein Is a Prognostic Marker of Transplant-Associated Coronary Artery Disease
Authors: Holvoet Paul , Van Cleemput Johan , Collen Désiré , Vanhaecke Johan
Published: June 2011

Abstract
—Retrospective studies identified oxidized low density lipoprotein (LDL) in the blood as a diagnostic marker of coronary artery disease (CAD). This prospective study sought to determine the prognostic value of oxidized LDL for CAD in cardiac transplant patients. Oxidized LDL was measured in 99 cardiac transplant patients with normal coronary angiograms at baseline and was measured again after a median follow-up of 2 years at the time of a second angiogram. Twenty-one patients developed angiographically detectable cardiac transplant vasculopathy (cases), and 78 individuals did not (controls). Cases had significantly higher baseline plasma levels of oxidized LDL than did controls: 1.18±0.70 versus 0.57±0.20 mg/dL (mean±SD,
P
<0.0001). The increase of oxidized LDL at the end of the follow-up was significantly higher in cases than in controls: 0.75±0.73 mg/dL versus 0.14±0.27 mg/dL (
P
<0.0001). Baseline levels of oxidized LDL predicted cardiac transplant vasculopathy (χ
2
=16,
P
<0.0001) independent of pretransplant ischemic cardiomyopathy, time after transplantation, age, and serum levels of LDL and high density lipoprotein cholesterol. The development of transplant CAD was associated with a further increase of plasma levels of oxidized LDL (χ
2
=14,
P
=0.0002). Oxidized LDL is a prognostic marker of transplant CAD.
Oxidized Cholesterol in the Diet Accelerates the Development of Atherosclerosis in LDL Receptor– and Apolipoprotein E–Deficient Mice
Authors: Staprans Ilona , Pan Xian-Mang , Rapp Joseph H. , Grunfeld Carl , Feingold Kenneth R.
Published: June 2011

Abstract
—The aim of the current study was to determine whether oxidized cholesterol in the diet accelerates atherosclerosis in low density lipoprotein receptor– (LDLR) and apolipoprotein E– (apo E) deficient mice. Mice were fed either a control diet or a diet containing oxidized cholesterol. For LDLR-deficient mice, the control diet consisted of regular mouse chow to which 1.0% cholesterol was added. The oxidized diet was identical to the control diet except that 5% of the added cholesterol was oxidized. In apo E–deficient mice, the control diet contained 0.15% cholesterol, whereas in the oxidized diet, 5% of the added cholesterol was oxidized. LDLR-deficient and apo E–deficient mice were fed the experimental diets for 7 and 4 months, respectively. In mice fed the oxidized-cholesterol diets, the levels of oxidized cholesterol in sera were increased. At the end of the experiment, aortas were removed and atherosclerosis was assessed. We found that in LDLR-deficient mice, feeding of an oxidized-cholesterol diet resulted in a 32% increase in fatty streak lesions (15.93±1.59% versus 21.00±1.38%,
P
<0.03). Similarly, in apo E–deficient mice, feeding of an oxidized-cholesterol diet increased fatty streak lesions by 38% (15.01±0.92% versus 20.70±0.86%,
P
<0.001). The results of the current study thus demonstrate that oxidized cholesterol in the diet accelerates fatty streak lesion formation in both LDLR- and apo E–deficient mice.
Ras/Rac-Dependent Activation of p38 Mitogen-Activated Protein Kinases in Smooth Muscle Cells Stimulated by Cyclic Strain Stress
Authors: Li Chaohong , Hu Yanhua , Sturm Gertraud , Wick Georg , Xu Qingbo
Published: June 2011

Abstract
—p38, a subfamily of the mitogen-activated protein kinases (MAPKs), is a crucial signal transducer between a variety of extracellular stimuli and gene expression in mammalian cells. This kinase is activated in cultured cells stimulated by heat shock, osmotic stress, and proinflammatory cytokines, but a similar activation of p38 MAPKs in vascular smooth muscle cells (SMCs) stimulated by mechanical stress has yet to be studied. We studied signal pathways leading to time- and strength-dependent p38 activation in rat SMCs in response to cyclic strain stress. p38 phosphorylation in stressed SMCs showed maximal activation at 10 minutes. This activation was significantly inhibited by pretreatment of the SMCs with pertussis toxin, a G-protein antagonist, and enhanced by treatment with suramin, a growth factor receptor antagonist, but opposite effects in the activation of extracellular signal–regulated kinases stimulated by mechanical forces were found. p38 activation was markedly reduced in stressed SMCs after protein kinase C depletion. Interestingly, SMC lines stably expressing dominant-negative ras (ras N17) or rac1 (rac1 N17) almost abolished p38 phosphorylation induced by cyclic strain stress. When p38 activation was inhibited by the specific inhibitor SB 202190, SMC migration, determined in a Boyden chamber in response to stimulation with platelet-derived growth factor-BB, and SMC proliferation, stimulated by cyclic strain stress, were abrogated. Thus, we provide the first evidence that cyclic strain stress rapidly activates p38 MAPKs via activation of protein kinase C ras/rac signal pathways, suggesting that p38 MAPKs are important signal transducers mediating the mechanical stress–induced cell responses essential for SMC migration and proliferation.
Lysophosphatidylcholine Inhibits Endothelial Cell Migration and Proliferation via Inhibition of the Extracellular Signal–Regulated Kinase Pathway
Authors: Rikitake Yoshiyuki , Kawashima Seinosuke , Yamashita Tomoya , Ueyama Tomomi , Ishido Satoshi , Hotta Hak , Hirata Ken-ichi , Yokoyama Mitsuhiro
Published: June 2011

Abstract
—Lysophosphatidylcholine (lysoPC), a major lipid component of oxidized low density lipoprotein, inhibits endothelial cell (EC) migration and proliferation, which are critical processes during angiogenesis and the repair of injured vessels. However, the mechanism(s) of lysoPC-induced inhibition of EC migration and proliferation has not been clarified. In this report, we demonstrate the critical role of extracellular signal–regulated kinase (ERK) in growth factor–stimulated EC migration and proliferation as well as their inhibition by lysoPC. EC migration and proliferation stimulated by basic fibroblast growth factor (FGF-2) were blocked by inhibition of ERK activity by both the specific mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 and the overexpression of a dominant-negative mutant of MEK1. Conversely, overexpression of a constitutively active mutant of MEK1 increased EC migration and proliferation, which were comparable to those of ECs stimulated with FGF-2. LysoPC inhibited FGF-2–induced ERK activation via prevention of Ras activation without inhibiting tyrosine phosphorylation of phospholipase C-γ. Taken together, our data demonstrate that ERK activity is required for FGF-2–induced EC migration and proliferation and suggest that inhibition of the Ras/ERK pathway by lysoPC contributes to the reduced EC migration and proliferation.
Apolipoprotein AII Enrichment of HDL Enhances Their Affinity for Class B Type I Scavenger Receptor but Inhibits Specific Cholesteryl Ester Uptake
Authors: Pilon Antoine , Briand Olivier , Lestavel Sophie , Copin Corinne , Majd Zouher , Fruchart Jean-Charles , Castro Graciela , Clavey Véronique
Published: June 2011

Abstract
—Apolipoproteins of high density lipoprotein (HDL) and especially apolipoprotein (apo)AI and apoAII have been demonstrated as binding directly to the class B type I scavenger receptor (SR-BI), the HDL receptor that mediates selective cholesteryl ester uptake. However, the functional relevance of the binding capacity of each apolipoprotein is still unknown. The human adrenal cell line, NCI-H295R, spontaneously expresses a high level of SR-BI, the major apoAI binding protein in these cells. As previously described for murine SR-BI, free apoAI, palmitoyl-oleoyl-phosphatidylcholine (POPC)-AI, and HDL are good ligands for human SR-BI. In vitro displacement of apoAI by apoAII in HDLs or in Lp AI purified from HDL by immunoaffinity enhances their ability to compete with POPC-AI to bind to SR-BI and also enhances their direct binding capacity. The next step was to determine whether the higher affinity of apoAII for SR-BI correlated with the specific uptake of cholesteryl esters from these HDLs. Free apoAII and, to a lesser extent, free apoAI that were added to the cell medium during uptake experiments inhibited the specific uptake of [
3
H]cholesteryl esters from HDL, indicating that binding sites on cells were the same as cholesteryl ester uptake sites. In direct experiments, the uptake of [
3
H]cholesteryl esters from apoAII-enriched HDL was highly reduced compared with the uptake from native HDL. These results demonstrate that in the human adrenal cell line expressing SR-BI as the major HDL binding protein, efficient apoAII binding has an inhibitory effect on the delivery of cholesteryl esters to cells.
Genetic Localization to Chromosome 1p32 of the Third Locus for Familial Hypercholesterolemia in a Utah Kindred
Authors: Hunt Steven C. , Hopkins Paul N. , Bulka Katrina , McDermott Michael T. , Thorne Thomas L. , Wardell Bryan B. , Bowen Benjamin R. , Ballinger Dennis G. , Skolnick Mark H. , Samuels Mark E.
Published: June 2011

Abstract
—Clinical familial hypercholesterolemia has been shown to result from mutations in 2 genes, the low density lipoprotein (LDL) receptor on chromosome 19 and apolipoprotein B on chromosome 2. However, we have recently described a Utah pedigree in which linkage to both genes was clearly excluded. A multipoint linkage analysis of 583 markers genotyped on 31 (18 affected) members of this pedigree was undertaken to localize a genetic region that may harbor a third gene that could result in clinical familial hypercholesterolemia. A multipoint log of the odds score of 6.8 was obtained for markers on 1p32. Haplotype carriers and affected status are completely concordant (18/18 persons). The phenotype is also expressed in young children (ages 4 and 9). Specific recombinant individuals in the pedigree restrict the region of linkage to an ≈17 cM interval between polymorphic markers D1S2130 and D1S1596. This region appears to overlap the region found linked to severe hypercholesterolemia in French and Spanish families. The identification of the gene in this region may provide important pathophysiological insights into new mechanisms that may lead to highly elevated LDL cholesterol and other associated dyslipidemic phenotypes.
Aging Influences Development and Progression of Early Aortic Atherosclerotic Lesions in Cholesterol-Fed Rabbits
Authors: Orlandi Augusto , Marcellini Marcella , Spagnoli Luigi Giusto
Published: June 2011

Abstract
—The arterial wall in aged animals shows an increased susceptibility to develop atherosclerotic lesions, although the mechanisms by which aging acts are still unclear. We investigated early aortic lesions in aged rabbits (5 to 6 years old, AH group) and young rabbits (2 months old, YH group) after 2 months of 0.2% cholesterol feeding. Fatty streaks or spots mainly in the proximal segments occupied a relative surface area that was greater in AH than in YH rabbits, although plasma cholesterol and lipoprotein levels did not differ. YH lesions showed an irregular endothelial profile mainly from accumulations of large, rounded, RAM 11–positive macrophagic foam cells. There was a higher percentage of myocytic, CD-5–positive, proliferating, and terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL)-positive cells and larger accumulation of glycosaminoglycans in AH fatty streaks than in YH lesions. Ligation-mediated polymerase chain reaction confirmed differences in apoptosis. Early fibromuscular coats and subendothelial plasma-like insudate were also observed in AH lesions. Aged-matched normocholesterolemic rabbits showed a diffuse aortic intimal thickening composed of myocytic cells with a synthetic phenotype and extracellular matrix rich in glycosaminoglycans. In addition, in aged rabbits, we observed a spontaneous increase of monocytes adhering to the endothelial surface and a reduced expression of endothelial nitric oxide synthase in areas distant from the branches. These plasma cholesterol–independent spontaneous changes in the aortic wall of aged rabbits seem to act as a multiple atherogenic risk factor. Moreover, age-related differences in the distribution, composition, and proliferative and apoptotic rates represent crucial events during the progression of early fatty streaks to advanced plaques.
Increased Flow and Shear Stress Enhance In Vivo Transforming Growth Factor-β1 After Experimental Arterial Injury
Authors: Song Ruo H. , Kocharyan Hrachya K. , Fortunato John E. , Glagov Seymour , Bassiouny Hisham S.
Published: June 2011

Abstract
—We have previously demonstrated that high-flow (HF) conditions inhibit experimental intimal hyperplasia. We hypothesized that such flow conditions may alter transforming growth factor-β1 (TGF-β1) after mural injury. The right common carotid artery (CCA) was balloon-injured in 54 New Zealand White male rabbits. Flow was thereafter preserved (normal flow [NF]), reduced by partial outflow occlusion (low flow [LF]), or increased by ligation of the left CCA (HF). Four sham-operated animals served as uninjured controls. Mean blood flow and pressure in the right CCA were measured before and after flow modulation and before euthanasia (3, 7, and 14 days). TGF-β1 mRNA and protein levels in the right CCA were determined by Northern and ELISA analyses at each time point. At 7 and 14 days, intimal hyperplasia was quantified, and the transmural localization of TGF-β1 was determined by immunohistochemical analysis. Mean flow was reduced from 22±1 to 10±3 mL/min in the LF group and increased to 34±2 mL/min in the HF group (
P
<0.001). Blood pressure was not different among the flow groups for all time points. Wall shear stress was markedly decreased in the LF group to 14±4 dyne/cm
2
and increased in the HF group to 63±6 dyne/cm
2
at 7 days compared with values in uninjured controls (39±2 dyne/cm
2
,
P
<0.001) and the NF group (44±7 dyne/cm
2
,
P
<0.001). At 14 days, wall shear stress was similar among the flow groups. The intima-to-media ratio was 5- and 2-fold greater in the LF group than in the HF and NF groups at 14 days. mRNA levels for TGF-β1 and its active ligand were increased in the HF group by at least 2- and 3-fold, respectively, at 3 and 7 days compared with levels in uninjured controls and the LF group (
P
<0.05) but were not different among the flow groups at 14 days. TGF-β1 preferentially localized in the abluminal vascular smooth muscle cells of the HF arterial segments. Flow- and shear-mediated release of TGF-β1 may therefore play a role in abrogating the proliferative and migratory response of vascular smooth muscle cells in the early stages after mural injury.
Insulin-Like Growth Factor–Binding Protein-3 Is Associated With the Presence and Extent of Coronary Arteriosclerosis
Authors: Schuler-Lüttmann Susanne , Mönnig Gerold , Enbergs Annette , Schulte Helmut , Breithardt Günter , Assmann Gerd , Kerber Sebastian , von Eckardstein Arnold
Published: June 2011

Abstract
—Aging is associated with the progression of arteriosclerosis and the decline of several endocrine functions. We therefore investigated the association of coronary arteriosclerosis with hormones, the serum concentrations of which change during aging. Coronary angiograms of 189 men <70 years old were evaluated by 3 semiquantitative score systems to estimate the extent of focal and diffuse vessel wall alterations. Fasting sera were analyzed for levels of glucose, lipids, thyroid-stimulating hormone, insulin, insulin-like growth factor I (IGF-I), IGF-binding protein-3 (IGFBP-3), dehydroepiandrosterone sulfate (DHEAS), testosterone, and sex hormone–binding globulin (SHBG). After adjustment for age, body mass index, and waist-to-hip ratio, 92 patients with ≥1 stenoses >70% differed from 97 patients without such focal lesions by higher serum levels of glucose, total and LDL cholesterol, and apolipoprotein (apo) B, as well as by lower serum levels of IGFBP-3. Multivariate analyses revealed significant and independent correlations of all 3 coronary scores with LDL cholesterol (or apoB) and IGFBP-3; of 2 coronary scores with age, glucose, and insulin; and of 1 score with IGF-I. No significant correlations existed for waist-to-hip ratio (or body mass index) and DHEAS (or testosterone or SHBG). IGFBP-3 explained 9% to 14% and 3.5% to 10% of the variances of focal and diffuse lesions, respectively. In conclusion, IGFBP-3 and, with much less strength and consistency, insulin and IGF-I, but not markers of hypothyroidism, adrenopause, and andropause, have statistically significant and independent associations with coronary arteriosclerosis in men.
Simultaneously Increased TxA
2
Activity in Isolated Arterioles and Platelets of Rats With Hyperhomocysteinemia
Authors: Ungvari Zoltan , Sarkadi-Nagy Eszter , Bagi Zsolt , Szollár Lajos , Koller Akos
Published: June 2011

Abstract
—We aimed to elucidate the effect of hyperhomocysteinemia (HHcy) on the synthesis of prostaglandins in rat skeletal muscle arterioles and platelets. Male Wistar rats were divided into 2 groups: (1) control rats, with plasma Hcy levels of 6.5±0.5 μmol/L (n=50) and (2) rats with HHcy, induced by daily intake of 1 g/kg body weight methionine in the drinking water for 4 weeks (plasma Hcy levels were 20.6±3.0 μmol/L,
P
<0.01 versus controls; n=50). Arterioles (diameter ≈130 μm) were isolated from the gracilis muscle, cannulated, and pressurized (at 80 mm Hg), and changes in their diameters were followed by video microscopy. Constrictions to bradykinin (BK; 10
−10
to 10
−7
mol/L) were significantly greater in HHcy than in control rat arterioles (at 10
−9
mol/L BK, changes were 11±3% in control and 41±9% in HHcy rats). The cyclooxygenase inhibitor indomethacin (10
−5
mol/L), the prostaglandin H
2
/thromboxane A
2
(PGH
2
/TxA
2
) receptor antagonist SQ 29,548 (10
−6
mol/L), or the TxA
2
synthase inhibitor furegrelate (5×10
−6
mol/L) significantly decreased constrictions to BK in both groups but more so in HHcy arterioles, thus eliminating the difference between responses of HHcy and control arterioles. Constrictions to U46619 (a TxA
2
analogue) were significantly greater in HHcy than in control arterioles (at 10
−8
mol/L U46619, values for controls were 33±2% and 54±3% for HHcy). Endothelium removal or indomethacin treatment attenuated constrictions to U46619 in HHcy arterioles and eliminated the difference in responses. Also, aggregation of platelets from HHcy rats to collagen and ADP was significantly enhanced compared with controls (with 5 μg/mL collagen: controls, 23±5%; HHcy, 49±5%; with 10
−7
mol/L ADP: controls, 25±3%; HHcy, 35±3%). Indomethacin or SQ 29,548 caused greater inhibition of aggregation of HHcy platelets compared with controls, thereby eliminating the differences between the 2 groups. Thus, HHcy enhances TxA
2
synthesis both in the arteriolar endothelium and platelets. By promoting vascular constriction and platelet aggregation simultaneously, these alterations are likely to contribute to the atherothrombotic vascular diseases described in HHcy.
Well-Defined Regions of Apolipoprotein B-100 Undergo Conformational Change During Its Intravascular Metabolism
Authors: Wang Xingyu , Pease Richard , Bertinato Jesse , Milne Ross W.
Published: June 2011

Abstract
—Apolipoprotein B (apoB)-100–containing lipoproteins are secreted from the liver as large triglyceride-rich very low density lipoproteins (VLDLs) into the circulation, where they are transformed, through the action of lipases and plasma lipid transfer proteins, into smaller, less buoyant, cholesteryl ester–rich low density lipoproteins (LDLs). As a consequence of this intravascular metabolism, apoB-containing lipoproteins are heterogeneous in size, in hydrated density, in surface charge, and in lipid and apolipoprotein composition. To identify specific regions of apoB that may undergo conformational changes during the intravascular transformation of VLDLs into LDLs, we have used a panel of 29 well-characterized anti-apoB monoclonal antibodies to determine whether individual apoB epitopes are differentially expressed in VLDL, intermediate density lipoprotein (IDL), and LDL subfractions isolated from 6 normolipidemic subjects. When analyzed in a solid-phase radioimmunoassay, the expression of most epitopes was remarkably similar in VLDLs, IDLs, and LDLs. Two epitopes that are close to the apoB LDL receptor–binding site show an increased expression in large (1.019 to 1.028 g/mL), medium (1.028 to 1.041 g/mL), and small (1.041 to 1.063 g/mL) LDLs compared with VLDLs and IDLs, and 2 epitopes situated between apoB residues 4342 and 4536 are significantly more immunoreactive in small and medium-sized LDLs compared with VLDLs, IDLs, and large LDLs. Therefore, as VLDL is converted to LDL, conformational changes identified by monoclonal antibodies occur at precise points in the metabolic cascade and are limited to well-defined regions of apoB structure. These conformational changes may correspond to alterations in apoB functional activities.
The A −844G Polymorphism in the PAI-1 Gene Is Associated With a Higher Risk of Venous Thrombosis in Factor V Leiden Carriers
Authors: Morange P. E. , Henry M. , Tregouët D. , Granel B. , Aillaud M. F. , Alessi M. C. , Juhan-Vague I.
Published: June 2011

Abstract
—Identification of combined genetic factors in factor V Leiden carriers is important for a more accurate risk assessment for venous thrombosis (VT). Among these individuals, we evaluated the role of polymorphisms of the plasminogen activator inhibitor-1 (PAI-1) gene in the thrombophilic phenotype. A total of 382 factor V Leiden carriers were included in the study. This population was divided into 3 groups. Group 1 (n=168) included individuals with a personal history of VT; group 2 (n=140) included individuals without personal VT but with a familial history of VT; and group 3 (n=74) included individuals without VT and with a fortuitous discovery of the factor V Leiden mutation. We compared the genotype distribution of 2 polymorphisms, A −844G and −675 4G/5G, located in the promoter region of the PAI-1 gene among these 3 groups of individuals. The A −844G allele frequency differed significantly among the 3 groups (
P
=0.048), the
A
allele being more frequent in patients who suffered from VT (61%) than in subjects without VT (52%,
P
=0.015), whereas no difference was observed between the 2 groups of asymptomatic individuals. The prevalence of genotype
AA
carriers was higher in patients with VT (38%) than in asymptomatic individuals (21%,
P
=0.015), leading to an odds ratio of 1.74 (95% confidence interval, 1.3 to 3.8). Carrying the
AA
genotype conferred a risk of deep VT of 2.08 (95% confidence interval, 1.28 to 3.40), whereas it did not seem to significantly influence the risk of pulmonary embolism. Concerning the −675 4G/5G polymorphism, no significant difference was observed among the 3 groups, the
4G
allele frequency being 0.54 (in group 1), 0.49 (in group 2), and 0.45 (in group 3). These data suggest a role for the −A844G PAI-1 gene polymorphism in the thrombophilic phenotype of factor V Leiden carriers.
Familial Thrombophilia Associated With Homozygosity for the Cystathionine β-Synthase 833T→C Mutation
Authors: Gaustadnes Mette , Rüdiger Niels , Rasmussen Karsten , Ingerslev Jørgen
Published: June 2011

Abstract
—Severe hyperhomocysteinemia due to cystathionine β-synthase (CBS) deficiency is a strong risk factor for premature cardiovascular disease. Among untreated patients, ≈50% have suffered a thromboembolic event by 30 years of age. We report on 3 sisters with severe hyperhomocysteinemia due to homozygosity for the CBS 833T→C mutation. These patients, who displayed no other known thrombophilic predisposition, had suffered single or multiple venous thrombosis before CBS deficiency was diagnosed relatively late in life. In this family, homozygosity for the 833T→C mutation was associated with a mild phenotype with respect to other sequelae of CBS deficiency. Consequently, our results indicate that most cases with this genotype may remain undiagnosed. Investigated family members heterozygous for the 833T→C mutation displayed normal total homocysteine in plasma (tHcy) levels, even when they were homozygous for the methylenetetrahydrofolate reductase 677C→T polymorphism. The prevalence of homozygosity for the 833T→C mutation has previously been estimated at no less than 1:20 500 in our population. Because a reduction of the severely elevated levels of tHcy in CBS deficiency reduces cardiovascular risk and because homozygosity for the 833T→C mutation is more prevalent than previously thought, our results emphasize the importance of measuring tHcy routinely in thrombophilia screening.
Oral, but Not Transdermal, Administration of Estrogens Lowers Tissue-Type Plasminogen Activator Levels in Humans Without Affecting Endothelial Synthesis
Authors: Giltay E. J. , Gooren L. J. G. , Emeis J. J. , Kooistra T. , Stehouwer C. D. A.
Published: June 2011

Abstract
—Oral estrogen administration decreases plasma levels of tissue-type plasminogen activator (tPA), which may be explained by a decrease in endothelial tPA synthesis, an increase in its hepatic clearance, or both. In the present study, we determined (1) differences between oral (ie, via the liver) ethinyl estradiol and transdermal (ie, systemic) 17β-estradiol administration on plasma antigen levels of tPA and plasminogen activator inhibitor type-1 before and after 4 months of hormone administration and (2) effects on endothelial tPA synthesis, by measuring the local increase in plasma tPA during venous occlusion of the upper extremity. Thirty transsexual males (median age 32 years, range 20 to 44 years ) were randomly assigned to either oral ethinyl estradiol (n=15) or transdermal 17β-estradiol (n=15); both treatments included the antiandrogen cyproterone acetate (CA). Ten males were treated with CA alone. Seventeen transsexual females (median age 27 years, range 18 to 37 years) were treated with intramuscular testosterone esters. Only oral ethinyl estradiol plus CA but neither transdermal 17β-estradiol plus CA, nor oral CA, nor parenteral testosterone lowered plasma tPA and plasminogen activator inhibitor-1 (
P
<0.001 for both). tPA release during venous occlusion was not affected by oral ethinyl estradiol plus CA in males (
P
=0.52) or by parenteral testosterone in females (
P
=0.89). These data are consistent with a previous observation, in rodents, that the decrease in tPA after oral estrogen administration can be explained by an increase in hepatic tPA clearance, leaving endothelial tPA synthesis unchanged, and suggest that these mechanisms also explain the decrease in tPA in humans.
Quest for Fire
Authors: Schultz David , Harrison David G.
Published: June 2011

Abstract
—There is an accumulating body evidence that atherosclerosis is either caused by or accompanied by oxidative events in the vessel wall. These oxidative events have been implicated in proatherogenic modification of proteins, alteration of gene expression, promotion of inflammation, remodeling of vessels, and perturbations of vascular tone. This body of literature has led to a dogma that oxidation is a prerequisite for the atherosclerotic process. In particular, oxidation of lipoproteins by activated macrophages in the subintimal space has been postulated to be an important early step in the atherosclerotic process.
Second-Generation Adenoviral Vectors Do Not Prevent Rapid Loss of Transgene Expression and Vector DNA From the Arterial Wall
Authors: Wen Shan , Schneider Darren B. , Driscoll Robert M. , Vassalli Giuseppe , Sassani André B. , Dichek David A.
Published: June 2011

Abstract
—The utility of adenoviral vectors for arterial gene transfer is limited by the brevity of their expression and by inflammatory host responses. As a step toward circumventing these difficulties, we used a rabbit model of in vivo arterial gene transfer to test 3 second-generation vectors: a vector containing a temperature-sensitive mutation in the E2A region, a vector deleted of E2A, and a vector that expresses the immunomodulatory 19-kDa glycoprotein (gp19k) from adenovirus 2. Compared with similar first-generation vectors, the second-generation vectors did not significantly prolong β-galactosidase transgene expression or decrease inflammation in the artery wall. Although cyclophosphamide ablated the immune and inflammatory responses to adenovirus infusion, it only marginally prolonged transgene expression (94% drop in expression between 3 and 14 days). In experiments performed with “null” adenoviral vectors (no transgene), loss of vector DNA from the arterial wall was also rapid (>99% decrease between 1 hour and 14 days), unrelated to dose, and only marginally blunted by cyclophosphamide. Thus, the early loss of transgene expression after adenoviral arterial gene transfer is due primarily to loss of vector DNA, is not correlated with the presence of local vascular inflammation, and cannot be prevented by use of E2A-defective viruses, expression of gp19k, or cyclophosphamide-mediated immunosuppression. Adenovirus-induced vascular inflammation can be prevented by cyclophosphamide treatment or by lowering the dose of infused virus. However, stabilization of adenovirus-mediated transgene expression in the arterial wall is a more elusive goal and will require novel approaches that prevent the early loss of vector DNA.
α
2
-Antiplasmin Gene Deficiency in Mice Does Not Affect Neointima Formation After Vascular Injury
Authors: Lijnen H. R. , Van Hoef B. , Dewerchin M. , Collen D.
Published: June 2011

Abstract
—The hypothesis that α
2
-antiplasmin (α
2
-AP), the main physiological plasmin inhibitor, plays a role in neointima formation was tested with use of a vascular injury model in wild-type (α
2
-AP
+/+
) and α
2
-AP–deficient (α
2
-AP
−/−
) mice. The neointimal and medial areas were similar 1 to 3 weeks after electric injury of the femoral artery in α
2
-AP
+/+
and α
2
-AP
−/−
mice, resulting in comparable intima/media ratios (eg, 0.43±0.12 and 0.42±0.11 2 weeks after injury). Nuclear cell counts in cross-sectional areas of the intima of the injured region were also comparable in arteries from α
2
-AP
+/+
and α
2
-AP
−/−
mice (78±19 and 69±8). Fibrin deposition was not significantly different in arteries of both genotypes 1 day after injury, and no mural thrombosis was detected 1 week after injury. Fibrinolytic activity in femoral arterial sections, as monitored by fibrin zymography, was higher in α
2
-AP
−/−
mice 1 week after injury (
P
<0.001) but was comparable in both genotypes 2 and 3 weeks after injury. Staining for elastin did not reveal significant degradation of the internal elastica lamina in either genotype. Immunocytochemical analysis revealed a comparable distribution pattern of α-actin–positive smooth muscle cells in both genotypes. These findings indicate that the endogenous fibrinolytic system of α
2
-AP
+/+
mice is capable of preventing fibrin deposition after vascular injury and suggest that α
2
-AP does not play a major role in smooth muscle cell migration and neointima formation in vivo.
Interferon-γ Induces Downregulation of Tangier Disease Gene (ATP-Binding-Cassette Transporter 1) in Macrophage-Derived Foam Cells
Authors: Panousis Constantinos G. , Zuckerman Steven H.
Published: June 2011

Abstract
—Cholesterol efflux is a fundamental process that serves to mitigate cholesterol accumulation and macrophage foam cell formation. Recently, we reported that cholesterol efflux to high density lipoprotein subfraction 3 was reduced by interferon-γ (IFN-γ) and that this decrease was associated with an increase in acyl coenzyme A:cholesterol acyltransferase (ACAT) expression. In the present study, although treatment of murine peritoneal macrophages with IFN-γ resulted in a 2-fold decrease in HDL-mediated cholesterol efflux, efflux to lipid-free apolipoprotein A-I was reduced >4-fold and approached basal levels. This decrease was associated with a 3- to 4-fold reduction in ATP-binding-cassette transporter 1 (ABC1) mRNA content, the gene responsible for the defect in Tangier disease. Consistent with the reduction in cholesterol and phospholipid efflux in Tangier fibroblasts, downregulation of ABC1 expression by IFN-γ also resulted in reduced phosphatidylcholine and sphingomyelin efflux to apolipoprotein A-I. Whereas foam cells had a 3-fold increase in ABC1 mRNA, the decrease in ABC1 message levels by IFN-γ was observed in foam cells and control macrophages. This effect of IFN-γ was independent of general macrophage activation (inasmuch as similar changes were not detected with granulocyte-macrophage colony–stimulating factor) and was not observed with other ABC transporters (inasmuch as the expression of the transporter in antigen processing was upregulated 4-fold in these same cells). Therefore, by decreasing cholesterol efflux through pathways that include the upregulation of ACAT and the downregulation of ABC1, IFN-γ can shift the equilibrium between macrophages and foam cells and thus impact the progression of an atherosclerotic lesion.
Differential Effect of National Cholesterol Education Program (NCEP) Step II Diet on HDL Cholesterol, Its Subfractions, and Apoprotein A-I Levels in Hypercholesterolemic Women and Men After 1 Year
Authors: Walden Carolyn E. , Retzlaff Barbara M. , Buck Brenda L. , Wallick Shari , McCann Barbara S. , Knopp Robert H.
Published: June 2011

Abstract
—We previously reported that high density lipoprotein cholesterol (HDL-C) decreases more in hypercholesterolemic (HC) women than in HC men ingesting an National Cholesterol Education Program (NCEP) Step II diet for 6 months. We examined these subjects to determine whether the differential HDL-C reduction persists after 12 months and whether it is associated with decreased HDL
2
-C and apoprotein A-I. Subjects were screened from an industrial workforce and were defined as HC if 2 low density lipoprotein cholesterol measurements were ≥75th percentile or defined as combined hyperlipidemic (CHL) if triglycerides were also ≥75th percentile. The subjects were then taught the NCEP Step II diet in 8 weekly classes and counseled quarterly. Seventy-three HC and 92 CHL women (mean ages 43 and 44 years, respectively) and 112 HC and 106 CHL men (ages 45 and 41 years, respectively) were studied. All groups reported similar total fat (24% to 26% kcal) and saturated fat (7.1% to 7.9% kcal) intakes at 1 year. HDL-C decreased 7.6% in HC women (
P
<0.01), exceeding the nonsignificant 1.3% decrease in HC men (
P
=0.000). HDL
2
-C decreased 16.7% in HC women (
P
<0.01) compared with the nonsignificant 0.5% increase in HC men (
P
=0.000). In CHL women and men, HDL-C decreased 3.5% and 3.9% (both
P
<0.01); HDL
2
-C decreased more in women (7.1%,
P
<0.01) than in men (4.3%, a nonsignificant difference). Apoprotein A-I decreased significantly (5.3%,
P
<0.01) in HC women only. Plasma triglycerides were unchanged. Low density lipoprotein cholesterol and weight changes were not different among the 4 groups. HDL-C, HDL
2
-C, and apoprotein A-I levels decreased more in HC women than in HC men after following the NCEP Step II diet for 1 year, continuing a trend observed with HDL-C at 6 months. The total HDL-C and HDL
2
-C reductions narrow the baseline differences between men and women by 50%. Whether this reduction impacts women’s protection from cardiovascular disease deserves future study. Nonetheless, the results point to sex-based differences in intrahepatic glucose and fatty acid metabolism linked to alterations in HDL formation and removal.
Physiological Concentration of 17β-Estradiol Retards the Progression of Severe Atherosclerosis Induced by a High-Cholesterol Diet Plus Balloon Catheter Injury
Authors: Hayashi Toshio , Jayachandran Muthuvel , Sumi Daigo , Thakur Navin Kumar , Esaki Teiji , Muto Emiko , Kano Hatsuyo , Asai Yukako , Iguchi Akihisa
Published: June 2011

Abstract
—The molecular mechanisms of the antiatherosclerotic effects of estrogen are not yet known. We evaluated the effects of 17β-estradiol (E
2
) on high cholesterol diet– (HCD; standard diet and 1% cholesterol) and balloon injury–induced atherosclerosis in female New Zealand White rabbits. The abdominal aortas of 40 oophorectomized (Groups 1 through 5) and 8 nonoophorectomized (Group 6) rabbits were injured by balloon catheter, and the animals were then divided into the following groups and treated for 10 weeks: Group 1, standard diet; Group 2, standard diet plus a moderate dose of E
2
(100 μg · kg
−1
· d
−1
); Group 3, HCD; Group 4, HCD plus a moderate dose of E
2
; Group 5, HCD plus a low dose of E
2
(20 μg · kg
−1
· d
−1
); and Group 6, HCD in nonoophorectomized rabbits. After the treatment phase, plasma E
2
was increased up to 282.2±45.5 pg/mL in Group 2, 263.0±41.5 pg/mL in Group 4, 87.9±18.8 pg/mL in Group 5, and 45.6±7.3 pg/mL in Group 6. HCD-mediated increases in plasma lipid levels were not changed by E
2
treatment, whereas E
2
decreased the aortic intimal thickening in Group 2 animals compared with those in Group 1 and reduced atherosclerosis in the thoracic and abdominal aortas of Group 4, 5, and 6 rabbits compared with those in Group 3. E
2
restored the impaired abdominal aortic endothelium–dependent relaxation of balloon-injured and HCD-supplemented rabbits, and E
2
increased basal nitric oxide (NO) release. The basal NO–releasing effect showed a significant, inverse relation with the severity of atherosclerosis. Plasma E
2
concentration also showed a significant, inverse relation with atherosclerotic area. In conclusion, physiological concentrations of E
2
can retard the progression of severe atherosclerosis and stabilize atheromas induced by HCD and balloon injury. The retardation may be partially mediated by endothelial NO function in vessels treated with E
2
.
Inhibition of Platelet Integrin α
IIb
β
3
by Peptides That Interfere With Protein Kinases and the β
3
Tail
Authors: Hers Ingeborg , Donath José , Litjens Pieter E. M. H. , van Willigen Gijsbert , Akkerman Jan-Willem N.
Published: June 2011

Abstract
—α-Thrombin stimulation of human platelets initiates inside-out signaling to integrin α
IIb
β
3
(glycoprotein IIb/IIIa), resulting in the exposure of ligand binding sites. In the present study, the regulation of α
IIb
β
3
via protein kinases was investigated in platelets permeabilized with streptolysin O by introducing peptides that interfere with these enzymes and with possible regulatory domains in the cytosolic tail of the β
3
subunit. Compared with intact platelets, the permeabilized platelets preserved >80% of the aggregation, secretion, and α
IIb
β
3
ligand binding capacity. The peptide YIYGSFK, a substrate for Src kinases, inhibited α-thrombin–induced ligand binding to α
IIb
β
3
, but a reversed peptide with Y→F substitutions (KFSGFIF) had no effect. Ligand binding to α
IIb
β
3
was also inhibited by the peptide RKRCLRRL, which binds irreversibly to the catalytic domain of protein kinase C. Peptides corresponding to parts of the protein C inhibitor and β
2
-glycoprotein I were used as negative controls and failed to interfere with ligand binding. Possible target domains for protein kinases are present in the cytoplasmic tail of the β
3
subunit. The LLITIHDR peptide, matching the membrane-proximal domain of β
3
(residues 717 to 724), had no effect, but NNPLYKEA (residues 743 to 750), EATSTFTN (residues 749 to 756), and TNITYRGT (residues 755 to 762), which mimicked overlapping domains of the carboxy-terminal part of β
3
, reduced α-thrombin–induced ligand binding by 60±4%, 97±1%, and 97±2% (n=3) at 500 μmol/L peptide, respectively. These observations indicate that Src kinases and protein kinase C take part in inside-out signaling to integrin α
IIb
β
3
and identify target domains in β
3
that contribute to the regulation of this integrin.
The Thromboxane Receptor Antagonist S18886 but Not Aspirin Inhibits Atherogenesis in Apo E–Deficient Mice
Authors: Cayatte Antonio J. , Du Yue , Oliver-Krasinski Jennifer , Lavielle Gilbert , Verbeuren Tony J. , Cohen Richard A.
Published: June 2011

Abstract
—Atherosclerosis involves a complex array of factors, including leukocyte adhesion and platelet vasoactive factors. Aspirin, which is used to prevent secondary complications of atherosclerosis, inhibits platelet production of thromboxane (Tx) A
2
. The actions of TxA
2
as well as of other arachidonic acid products, such as prostaglandin (PG) H
2
, PGF
2α
, hydroxyeicosatetraenoic acids, and isoprostanes, can be effectively antagonized by blocking thromboxane (TP) receptors. The purpose of this study was to determine the role of platelet-derived TxA
2
in atherosclerotic lesion development by comparing the effects of aspirin and the TP receptor antagonist S18886. The effect of 11 weeks of treatment with aspirin (30 mg · kg
−1
· d
−1
) or S18886 (5 mg · kg
−1
· d
−1
) on aortic root atherosclerotic lesions, serum levels of intercellular adhesion molecule-1 (ICAM-1), and the TxA
2
metabolite TxB
2
was determined in apolipoprotein E–deficient mice at 21 weeks of age. Both treatments did not affect body or heart weight or serum cholesterol levels. Aspirin, to a greater extent than S18886, significantly decreased serum TxB
2
levels, indicating the greater efficacy of aspirin in preventing platelet synthesis of TxA
2
. S18886, but not aspirin, significantly decreased aortic root lesions as well as serum ICAM-1 levels. S18886 also prevented the increased expression of ICAM-1 in cultured human endothelial cells stimulated by the TP receptor agonist U46619. These results indicate that inhibition of platelet TxA
2
synthesis with aspirin has no significant effect on atherogenesis or adhesion molecule levels. The effects of S18886 suggest that blockade of TP receptors inhibits atherosclerosis by a mechanism independent of platelet-derived TxA
2
, perhaps by preventing the expression of adhesion molecules whose expression is stimulated by eicosanoids other than TxA
2
.
Multiple Connexin Expression in Regenerating Arterial Endothelial Gap Junctions
Authors: Yeh Hung-I , Lai Yu-Jun , Chang Hao-Min , Ko Yu-Shien , Severs Nicholas J. , Tsai Cheng-Ho
Published: June 2011

Abstract
—Endothelial cells form gap junctions that, according to vessel type, may be composed of up to 3 types of connexin, connexin37, connexin40, and connexin43. Although changes in connexin expression have been linked to growth and injury in cultured endothelial cells, information on connexin expression in regenerating endothelium in situ is lacking. We investigated gap junction distribution and expression of all 3 endothelial connexins during healing in rat carotid artery after denudation injury. En face viewing of the vascular luminal surface by means of immunoconfocal microscopy was used to examine the spatial and temporal expression pattern of the endothelial connexins. Gap junction spots labeled by specific antisera against connexin37, connexin40, and connexin43 were quantified 7, 14, and 28 days after injury, and the relations among the connexins were examined by using colocalization analysis. Complementary electron microscopy was also conducted. After injury, the regenerating endothelium initially expressed small, sparse gap junctions, the numbers of which progressively increased to values equivalent to those of controls. Although connexin40 gap-junctional spot size and area returned to uninjured levels by 28 days after injury, connexin37 and connexin43 spot size and area exceeded those of the uninjured artery (
P
<0.05). Double-label analysis showed that even though colocalization of connexins to the same gap-junctional spot is a common feature, the extent of colocalization was time dependent (>80% in the intact artery at postinjury day 28 and <70% at postinjury days 7 and 14,
P
<0.01). We conclude that distinct alterations in expression of the 3 connexins are associated with regeneration of the arterial endothelium in situ, implying different intercellular communication requirements during the various phases of the healing process.
A Functional Polymorphism in the Promoter Region of the Microsomal Triglyceride Transfer Protein (MTP −493G/T) Influences Lipoprotein Phenotype in Familial Hypercholesterolemia
Authors: Björn Lundahl  , Leren Trond P. , Ose Leiv , Hamsten Anders , Karpe Fredrik
Published: June 2011

Abstract
—The microsomal triglyceride transfer protein (MTP) has a key function in intracellular apolipoprotein (apo) B lipidation and secretion of very low density lipoprotein (VLDL). A recently discovered functional polymorphism in the promoter of the MTP gene (−493G/T) affects the plasma concentration of low density lipoprotein (LDL) cholesterol and the VLDL distribution between large and small particle species in healthy men. This phenotype is likely to be explained by an effect on VLDL synthesis. Against this background, we studied the effect of the MTP−493G/T polymorphism in a large cohort (217 men and 211 women) with heterozygous familial hypercholesterolemia (FH). A 40% to 50% lower serum triglyceride level was observed in homozygous carriers of the MTP−493
T
allele (T/T, 0.93±0.34; G/T, 1.54±1.40; and G/G, 1.56±1.24 mmol/L; T/T vs G/T
P
=0.04, T/T vs G/G
P
=0.02). In contrast to the situation in healthy subjects, the MTP promoter polymorphism did not have a significant effect on the LDL cholesterol levels in FH subjects, although the same trend was observed (T/T, 7.31±1.87; G/T, 7.80±2.12; and G/G, 7.91±2.31 mmol/L, NS). Adjustment for the apo E gene polymorphism by inclusion of subjects homozygous for the apo E3 allele only revealed a reciprocal high density lipoprotein cholesterol–elevating effect (T/T, 1.41±0.73; G/T, 1.18±0.27; and G/G, 1.16±0.29 mmol/L; T/T vs G/T
P
=0.06, T/T vs G/G
P
=0.04). This effect seemed to be sex-specific because it was accounted for by the female patients. In conclusion, the LDL cholesterol–lowering effect of the rare MTP gene promoter variant (MTP−493T) present in healthy subjects is shifted to a triglyceride-lowering effect in FH. These data suggest that the MTP gene has a role in modulating the clinical phenotype of FH.