bluuebunny/crossref_metadata_2025 huggingface dataset:
A Common Mutation in the Lipoprotein Lipase Gene Promoter, −93T/G, Is Associated With Lower Plasma Triglyceride Levels and Increased Promoter Activity In Vitro
Authors: Hall Stephen , Chu Grace , Miller George , Cruickshank Kennedy , Cooper Jaqueline A. , Humphries Steve E. , Talmud Philippa J.
Published: June 2011

Abstract
Single-strand conformational polymorphism analysis of the lipoprotein lipase promoter identified a T→G transition at position −93. The frequency in healthy white men was 3.4% (n=1575). There was an 83% allelic association between −93T→G and Asp
9
→Asn (D9N); all N9 mutations occurred on a −93G allele, but not all −93G mutations occurred on an N9 allele. It was thus possible to assess the effect on plasma triglyceride (Tg) levels of the rare −93G mutation in the presence of the wild-type D9. Carriers of the −93G, with genotype TG/DD, had significantly lower Tg levels than TT/DD individuals (1.36 versus 1.78 mmol/L,
P
=.01); carriers of both mutations (TG/DN) had the highest Tg levels (1.93 mmol/L). When the group was stratified above and below the sample mean for body mass index (BMI), carriers of the −93G on a D9 allele (TG/DD) were “protected” against the Tg-raising effect of obesity, as assessed by BMI. In Afro-Caribbeans (n=91), the carrier frequency of −93G was 18-fold higher (63%), with weaker (17%) allelic association between −93G and N9. In vitro, the −93G promoter had 24% higher activity than the −93T in a rat smooth muscle cell line and 18% higher activity in a human adrenal cell line. A protein identified by band-shift assays bound to the −93G but not to the −93T allele, which may explain the lower Tg levels in −93G carriers.
Role of Tyrosine Kinases in Extracellular Matrix–Mediated Modulation of Arterial Smooth Muscle Cell Phenotype
Authors: Hedin Ulf , Thyberg Johan , Roy Joy , Dumitrescu Alexandra , Tran Phan Kiet
Published: June 2011

Abstract
Fibronectin (FN) promotes the modulation of freshly isolated arterial smooth muscle cells (SMCs) from a contractile to a synthetic phenotype by interacting with integrins on the cell surface. This process is characterized by a structural and functional transformation of the cells, including a reorganization of the cytoskeleton, the formation of a large secretory apparatus, and the acquisition of proliferative capacity. In this study we have investigated the role of integrin signaling through tyrosine kinases in the structural changes that occur in SMCs during primary culture on FN. A gradual increase in phosphotyrosine staining in focal adhesions and a concomitant increase in tyrosine phosphorylation of proteins including focal adhesion kinase were observed. In contrast, cells seeded on laminin formed few focal adhesions, and tyrosine phosphorylation of proteins was less than in cells cultured on FN. Treatment of cells cultured on FN with the tyrosine kinase inhibitor genistein strongly suppressed focal adhesion formation, cell spreading, and cytoskeletal reorganization. In addition, electron microscopic analysis demonstrated that the phenotypic modulation was slowed down. These results indicate that the ability of extracellular matrix components to promote a change in the phenotypic properties of SMCs depends on the assembly of focal adhesions with associated tyrosine kinase activity.
Altered Platelet Function Detected by Flow Cytometry
Authors: Knight Charles J. , Panesar Manvinder , Wright Christine , Clarke Debbie , Butowski Paul S. , Patel Deven , Patrineli Alexandra , Fox Kim , Goodall Alison H.
Published: June 2011

Abstract
Platelet activation state and responsiveness to physiological agonists were measured in 65 patients with documented coronary artery disease (54 male and 11 female; mean age, 58 years). Twelve patients (mean age, 52 years), selected at random from the male cohort, were compared with 12 age-matched male control subjects (mean age, 52 years) and with 10 normal, young male subjects (mean age, 25 years). Whole-blood flow cytometry was used to measure platelet activation status ex vivo and platelet responsiveness to physiological agonists in vitro. Peripheral blood samples were analyzed for bound fibrinogen and expression of P-selectin, GPIb, and GPIIb-IIIa at rest and in response to ADP (0.1 to 10 μmol/L) and thrombin (0.02 to 0.32 μ/mL). No significant differences were seen in the basal levels of fibrinogen binding between any of the groups, but P-selectin expression was significantly lower in patients compared with age-matched control subjects (
P
=.0005). When stimulated with agonists, patients’ platelets had significantly decreased fibrinogen binding (
P
<.03) but no difference in P-selectin expression compared with the age-matched group. Both agonist-induced fibrinogen binding and P-selectin expression were, however, higher in the young subjects compared with either the older control group or the patients (
P
<.05). GPIb and GPIIb-IIIa expression were lowest in the patients with angina and highest in the young control subjects, with levels in the age-matched control subjects falling between these values. Data from the total patient cohort (n=65) were identical to those in the smaller cohort (n=12). In conclusion, atherosclerosis impairs platelet aggregatory responses (fibrinogen binding) over and above the decreased response seen with age. Platelet degranulation (P-selectin expression) is also impaired in patients with coronary artery disease, but only in comparison with younger subjects, not age-matched controls.
Homocysteine as a Risk Factor for Vascular Disease
Authors: Majors Alana , Ehrhart L. Allen , Pezacka Ewa H.
Published: June 2011

Abstract
An increased plasma homocysteine level is an independent risk factor for vascular disease. However, the pathological mechanisms by which homocysteine promotes atherosclerosis are not yet clearly defined. Arterial smooth muscle cells cultured in the presence of homocysteine grew to a higher density and produced and accumulated collagen at levels significantly above control values. Homocysteine concentrations as low as 50 μmol/L significantly increased both cell density and collagen production. Cell density increased by as much as 43% in homocysteine-treated cultures. Homocysteine increased collagen production in a dose-dependent manner. Smooth muscle cells treated with homocysteine at concentrations observed in patients with hyperhomocysteinemia had collagen synthesis rates as high as 214% of control values. Likewise, collagen accumulation in the cell layer was nearly doubled in homocysteine-treated cultures. Addition of aquacobalamin to homocysteine-treated cultures controlled the increase in smooth muscle cell proliferation and collagen production. These results indicate a cellular mechanism for the atherogenicity of homocysteine and provide insight into a potential preventive treatment.
Risk of Coronary Heart Disease and Activation of Factor XII in Middle-Aged Men
Authors: Miller G.J. , Esnouf M.P. , Burgess A.I. , Cooper J.A. , Mitchell J.P.
Published: June 2011

Abstract
Increased activity is known to be present in the extrinsic, intrinsic, and final common pathways of the hemostatic system in men at high risk of coronary heart disease (CHD), but the status of the contact system of coagulation in this condition is uncertain. Plasma levels of activated factor XII (XIIa), the initial product of contact activation, have therefore been measured by ELISA in 2464 men aged 51 to 62 years, clinically free of CHD, who were taking part in a prospective cardiovascular survey based in general medical practices. Statistically significant, independent, and positive associations of XIIa were found with serum cholesterol and triglyceride concentrations, blood pressure, body mass index, factor VII activity, plasma fibrinogen concentration, and tobacco smoking, all associated with CHD. Plasma XIIa also increased with recent alcohol intake. Men in the highest quintile of risk according to their conventional risk factors had a mean XIIa of 2.07 ng/mL (95% confidence interval 1.99-2.16), 31% higher than that of men in the lowest quintile (1.58; 95% confidence interval 1.51-1.65). Thus, the contact system of coagulation appears to be activated when CHD risk is increased. Furthermore, the independent associations of XIIa with the major conventional CHD risk factors and its broad range of values in the general population (0.1 to 12.5 ng/mL), combined with a relatively low day-to-day variability in individuals (the within-person component of its total variation being 14.7%), suggest its potential usefulness as a marker of atherosclerotic vascular damage.
HDL and ApoA Prevent Cell Death of Endothelial Cells Induced by Oxidized LDL
Authors: Suc Isabelle , Escargueil-Blanc Isabelle , Troly Muriel , Salvayre Robert , Nègre-Salvayre Anne
Published: June 2011

Abstract
We have previously demonstrated that toxic doses of mildly oxidized LDL evokes in cultured cells a delayed and sustained rise of cytosolic [Ca
2+
], eliciting in turn irreversible cell damage and leading finally to cell death. HDL and delipidated apolipoprotein (apo) A prevented effectively the toxic effect of oxidized LDL to bovine aortic endothelial cells, in a time- and dose-dependent manner. The major part of the protective effect was mimicked by purified apoA-I, whereas purified apoA-II exhibited only very low protective activity. The protective effect was independent of the paraoxonase-linked HDL activity. The protective effect of HDL is independent of the contact of HDL with oxidized LDL, as shown by preincubation of oxidized LDL with HDL or apoA. In contrast, the protective effect was dependent on the integrity of apoA and on the contact of HDL with cells, thus suggesting that HDL acts directly on cells by enhancing their resistance against oxidized LDL. Preincubation experiments show that the protective effect is dependent on the duration of the contact of cells with HDL (maximal effect observed after 12 to 16 hours’ preincubation), is also dependent on protein synthesis, and is persistent for at least 48 hours after the end of the contact of HDL with cells. Finally, effective concentrations of HDL inhibit the Ca
2+
peak, which is directly involved in the cytotoxic effect of oxidized LDL, as shown by the inhibitory effect of Ca
2+
chelators. All together, these results suggest that HDL, mainly apoA-I, increases the resistance of endothelial cells against oxidized LDL and prevents its toxic (apoptotic) effect by blocking the pathogenic intracellular signaling (culminating in sustained Ca
2+
rise) involved in cell death.
Altered Compliance and Residual Strain Precede Angiographically Detectable Early Atherosclerosis in Low-Density Lipoprotein Receptor Deficiency
Authors: Hong Mun K. , Vossoughi Jafar , Mintz Gary S. , Kauffman Richard D. , Hoyt Robert F. , Cornhill J. Fredrick , Herderick Edward E. , Leon Martin B. , Hoeg Jeffrey M.
Published: June 2011

Background
This study was performed to detect changes in vascular biomechanical properties early in atherogenesis.

Methods and Results
Age- and weight-matched LDL-receptor deficient Watanabe hypercholesterolemic male rabbits (Group I: n=11) and normal rabbits (Group II: n=11) were studied. Fasting plasma lipoprotein concentrations, aortic angiography and intravascular ultrasound, in vivo aortic compliance evaluation, ex vivo aortic residual strain measurements, aortic lipid content and histopathology were determined. Plasma cholesterol was increased 9.8 fold and aortic cholesterol content was increased from 20 to 43 fold in Group I compared to Group II, respectively (
P
<.00005). Angiography revealed no stenoses in either group, whereas intravascular ultrasound and histological studies of Group I showed small circumferential plaques with <10% cross-sectional area involvement. The residual strain in Group I was significantly increased in the ascending thoracic aorta (22.1±6.9% versus 10.4±3.2% in Group II,
P
<.0001), descending thoracic aorta (15.7±7.2% versus 4.8±1.3% in Group II,
P
<.0001), and abdominal aorta (18.0±4.8% versus 8.3±6.3% in Group II,
P
<.005). Changes in residual strain were inversely correlated with the aortic cholesterol content in the ascending thoracic aorta (
r
=−.72;
P
=−.001), descending thoracic aorta (
r
=−.95;
P
<.001), and abdominal aorta (
r
=−.51;
P
=.019).

Conclusions
Early atherosclerosis in LDL-receptor deficient rabbits, undetectable by angiography yet observed by intravascular ultrasound imaging and histology, is associated with marked changes in ex vivo residual strain. Alterations in vascular biomechanical properties, associated with changes in cholesterol content, may have physiologic consequences and may be useful in detecting and quantitating early atherosclerosis.
Increased Blood Flow Induces Regression of Intimal Hyperplasia
Authors: Mattsson Erney J. R. , Kohler Ted R. , Vergel Selina M. , Clowes Alexander W.
Published: June 2011

Abstract
We have previously shown that high shear stress inhibits growth of developing neointima in a primate model of polytetrafluoroethylene (PTFE) graft healing. We used this model to test the hypothesis that increased shear stress can cause atrophy of an established neointima. High porosity PTFE grafts were inserted into the aorto-iliac circulation bilaterally in baboons. These grafts develop neointimal hyperplasia comprising smooth muscle cells and a luminal surface of confluent endothelium. Neointima was allowed to develop for 2 months. At that time 8 animals were sacrificed. In eight other animals blood flow in one of two grafts was increased by construction of a femoral arterio-venous fistula. These animals were sacrificed 2 months later (4 months after graft placement). At four months, intimal cross sectional area was smaller on the high shear stress side compared to the contralateral, normal shear stress side (2.53±0.75 versus 6.83±0.65 mm
2
,
P
<.05). Neointima from grafts exposed to 2 months normal shear stress followed by 2 months of high shear stress had regressed when compared to normal-shear stress grafts studied at 2 months (2.53±0.75 versus 4.56±0.68 mm
2
,
P
<.05). Morphometric analysis using transmission electron microscopy revealed that the decrease in intimal cross sectional area was attributable to a loss of both smooth muscle cells and matrix. Endothelial nitric oxide synthase was induced in high-flow graft intima. These observations support the conclusion that elevated shear stress can cause vessel wall atrophy. This process might be mediated by nitric oxide.
β-VLDL Hypercholesterolemia Relative to LDL Hypercholesterolemia Is Associated With Higher Levels of Oxidized Lipoproteins and a More Rapid Progression of Coronary Atherosclerosis in Rabbits
Authors: Holvoet Paul , Collen Désiré
Published: June 2011

Abstract
The accumulation of the oxidized apolipoprotein, apoB-100, containing lipoproteins in the arterial wall and the progression of coronary atherosclerotic lesions in rabbits with β-VLDL and LDL hypercholesterolemia was compared. In New Zealand White (NZW) rabbits on a 0.125% cholesterol diet, LDL cholesterol levels increased from 14±1 mg/dL (mean±SEM; n=9) to 170±34 mg/dL (n=10,
P
=.0002). On 0.5% cholesterol, LDL cholesterol levels were similar, but β-VLDL cholesterol levels increased from 60±4 mg/dL (n=10) to 550±75 mg/dL (n=8;
P
<.0001). In Watanabe heritable hyperlipidemic (WHHL) rabbits, LDL cholesterol levels were 2.3 -fold higher (n=13;
P
<.0001) than in NZW rabbits on 0.5% cholesterol, whereas their β-VLDL cholesterol levels were 3.7-fold lower (
P
<.0001), resulting in similar total cholesterol levels. At 2 months, mean intimal areas of lesions in the coronary arteries of NZW rabbits on 0.125% cholesterol were 0.13±0.045 mm
2
(n=4; mean±SEM) and were 5.8-fold (n=4;
P
=.016) and 2.0-fold (n=6;
P
=NS versus 0.125% cholesterol and
P
=.014 versus 0.5% cholesterol) higher in NZW rabbits on 0.5% cholesterol and in WHHL rabbits, respectively. At 5 months, mean intimal areas were 0.47±0.088 mm
2
(n=6) in NZW rabbits on 0.125% cholesterol and were 4.5-fold (n=4;
P
=.0001) and 2.0-fold (n=7;
P
=.012 and
P
=.0019) higher in rabbits on 0.5% cholesterol and in WHHL rabbits, respectively. Levels of oxidized apoB-100 containing lipoproteins (both β-VLDL and LDL) in the lesions correlated with mean intimal area (
r
=.88; n=31;
P
<.0001) of those lesions and with the plasma levels of total β-VLDL/LDL (
r
=.72;
P
<.0001). Levels of oxidized apoB-100 containing lipoproteins in the arterial wall correlate with progression of hypercholesterolemia- induced coronary atherosclerotic lesions. Plasma levels of β-VLDL relative to similar increases in LDL result in a more pronounced accumulation of oxidized apoB-100 containing lipoproteins in the arterial wall and in the plasma and a more rapid progression of coronary atherosclerosis.
Quantitation of Platelet-Derived Growth Factor Receptors in Human Arterial Smooth Muscle Cells In Vitro
Authors: Krettek Alexandra , Fager Gunnar , Jernberg Peter , Östergren-Lundén Gunnel , Lustig Florentyna
Published: June 2011

Abstract
Platelet-derived growth factor (PDGF) is suggested to play an important role in the development of atherosclerosis as a migratory and mitogenic stimulus to arterial smooth muscle cells (ASMCs). Stimulated and unstimulated ASMCs were studied with respect to PDGF receptor (PDGF-R) mRNA and protein expression. Quantitative RT-PCR was developed for simultaneous evaluation of both PDGF-Rα and -Rβ mRNA expression and a quantitative ELISA for estimation of corresponding PDGF-R subunits. On the mRNA level, the overall PDGF-Rβ expression was approximately 100 times lower than that of PDGF-Rα. Furthermore, although PDGF-Rα mRNA levels were high irrespective of hASMC phenotype, PDGF-Rβ mRNA was influenced by serum stimulation with lower copy numbers in proliferating and confluent cells compared with quiescent cells. On the protein level, quiescent hASMCs expressed 10 times more PDGF-Rβ than PDGF-Rα. Serum stimulation decreased cell surface PDGF-Rs, with most prominent loss of PDGF-Rα (ELISA and immunohistochemistry). Our results suggest a differential regulatory pattern for PDGF-Rα and -Rβ and are compatible with the usage of alternative promoters for regulation of -Rα expression. Further, it seems that the number of available receptor subunits is not the only determinant of variations in cell stimulation with different PDGF isoforms.
Correction of Hypertriglyceridemia and Impaired Fat Tolerance in Lipoprotein Lipase–Deficient Mice by Adenovirus-Mediated Expression of Human Lipoprotein Lipase
Authors: Excoffon Katherine J. D. Ashbourne , Liu Guoqing , Miao Li , Wilson Janet E. , McManus Bruce M. , Semenkovich Clay F. , Coleman Trey , Benoit Patrick , Duverger Nicolas , Branellec Didier , Denefle Patrice , Hayden Michael R. , Lewis M. E. Suzanne
Published: June 2011

AbstractHumans homozygous or heterozygous for mutations in the lipoprotein lipase (LPL) gene demonstrate significant disturbances in plasma lipoproteins, including raised triglyceride (TG) and reduced HDL cholesterol levels. In this study we explored the feasibility of adenovirus-mediated gene replacement therapy for LPL deficiency. A total of 5×109plaque-forming units (pfu) of an E1/E3–deleted adenovirus expressing either human LPL (Ad-LPL) or the bacterial β-galactosidase gene (Ad-LacZ) as a control were administered to mice heterozygous for targeted disruption in the LPL gene (n=57). Peak expression of total postheparin plasma LPL activity was observed at day 7 in Ad-LPL mice versus Ad-LacZ controls (834±133 vs 313±89 mU/mL,P<.01), and correlated with human-specific LPL activity (522±219 mU/mL) and mass (9214±782 ng/mL), a change that was significant to 14 and 42 days, respectively. At day 7, plasma TGs were significantly reduced relative to Ad-LacZ mice (0.17±0.07 vs 1.90±0.89 mmol/L,P<.01) but returned to endogenous levels by day 42. Ectopic liver expression of human LPL was confirmed by in situ hybridization analysis and from raised LPL activity and mass in liver homogenates. Analysis of plasma lipoprotein composition revealed a marked decrease in VLDL-derived TGs. Severely impaired oral and intravenous fat-load tolerance in LPL-deficient mice was subsequently corrected after Ad-LPL administration and closely paralleled that observed in wild-type mice. These findings suggest that liver-targeted, adenovirus-mediated LPL gene transfer offers an effective means for transient correction of altered lipoprotein metabolism and impaired fat tolerance due to LPL deficiency.
Contribution of Factor VII Genotype to Activated FVII Levels
Authors: Bernardi F. , Arcieri P. , Bertina R. M. , Chiarotti F. , Corral J. , Pinotti M. , Prydz H. , Samama M. , Sandset P. M. , Strom R. , Garcia V. Vicente , Mariani G.
Published: June 2011

AbstractThe relationship between coagulation factor VII (FVII) levels in plasma and FVII genotypes, determined by three polymorphisms (5′F7, IVS7, and 353R/Q), were studied in 500 control subjects enrolled in a European multicenter study. The selection of particular FVII genotypes and the analysis of variance clearly indicated the independent contribution of a single 5′F7 insertion (A2) or 353Q (M2) allele to lowering plasma levels of activated FVII (FVIIa) (by a mean 25%). The M2 allele alone was found to make a major contribution to the genetically determined component of the FVIIa levels. Genotypes associated with low FVII levels were significantly rarer in the northern part of Europe (Oslo) than in the southern part (Rome, Murcia). The contribution made by the FVII genotype to the total variance of FVIIa levels was higher (30%) than that made to either FVII activity (25%) or FVII antigen (12%). Subjects with different FVII genotypes showed up to fivefold differences in mean FVIIa values, thus allowing attribution of a substantial part of the considerable interindividual variation to genetic variation, which may be of assistance in the interpretation of FVIIa levels on an individual basis. When FVII levels were adjusted by age and by triglyceride levels, the contribution of FVII genotypes to the FVII phenotypic variance was virtually unchanged. Taken together, these data indicate that in healthy control subjects the FVII genotype is a major predictor of plasma FVIIa levels and would support further study on the role of FVII genetic components in the development of cardiovascular disease.
Thrombin Stimulates Syndecan-1 Promotor Activity and Expression of a Form of Syndecan-1 That Binds Antithrombin III in Vascular Smooth Muscle Cells
Authors: Cizmeci-Smith Gunay , Carey David J.
Published: June 2011

Abstract
Vascular smooth muscle (VSM) cells express transmembrane proteoglycans of the syndecan gene family. We reported previously that the expression of syndecans by VSM cells is regulated by mitogens such as serum, platelet-derived growth factor, and basic fibroblast growth factor and that syndecan expression is induced after balloon injury in vivo. We now show that thrombin is a potent inducer of syndecan-1 expression in VSM cells. Transient transfection experiments with a rat syndecan-1 promoter construct demonstrated that thrombin stimulates transcription of the syndecan-1 gene. Syndecan expression in response to thrombin was not inhibited by downregulation of protein kinase C. Thrombin-induced syndecan-1 expression was dependent on tyrosine kinase activity. Calcium was necessary for syndecan-1 expression, but increasing the intracellular calcium levels was not sufficient to induce syndecan-1 expression. Analysis of antithrombin III (AT III) binding activity revealed that thrombin caused an increase in the synthesis of syndecan-1 molecules that exhibited high-affinity AT III binding. These results suggest that VSM cells could play an important role in controlling local thrombus formation subsequent to vascular injury, via a feedback mechanism that involves thrombin-induced stimulation of an inhibitor of thrombin activity.
High Prevalence of Hyperhomocysteinemia and Asymptomatic Vascular Disease in Siblings of Young Patients With Vascular Disease and Hyperhomocysteinemia
Authors: de Jong Sylvia C. , Stehouwer Coen D. A. , Mackaay Albert J. C. , van den Berg Michiel , Bulterijs Ellen J. , Visser Frans C. , Bax Jeroen , Rauwerda Jan A.
Published: June 2011

Abstract
Hyperhomocysteinemia (HHC) is associated with an increased risk of atherosclerotic vascular disease and may be inherited. Fasting and postmethionine HHC are independent risk factors that overlap to a limited extent. To study the familial occurrence of HHC, we investigated the prevalence of HHC (both fasting and after methionine) among 450 siblings of 167 consecutive young patients with vascular disease and postmethionine HHC. Furthermore, all subjects with postmethionine HHC (n=125) were invited for noninvasive vascular testing; 101 (80.8%) agreed. Of those with a normal postmethionine plasma level (n=325), we randomly selected 73 subjects for further studies; 53 agreed (72.6%). Thus, a total of 154 siblings underwent ultrasonography of the carotid arteries, measurement of ankle-brachial pressure indices at rest and after a treadmill exercise test, and exercise electrocardiographic stress testing. We observed HHC after methionine, fasting, or both, in 27.8% (95% CI, 23.7 to 31.9), 11.1% (CI, 8.2 to 14.0) and 8.7% (CI, 6.1 to 11.3) of the siblings. Abnormal peripheral, coronary, or carotid artery tests were observed in 35.7% (CI, 28.1 to 43.3), 7.1% (CI, 3.0 to 11.2), and 7.1% (CI, 3.0 to 11.2). Univariate and multivariate analyses revealed weak evidence of a relationship with homocysteine levels.

In conclusion, we found a high prevalence of HHC and asymptomatic vascular disease in siblings of young patients with vascular (mainly peripheral arterial) disease and HHC. Our data raise the possibility that homocysteine does not play a major role in the early, asymptomatic phases of vascular disease, at least among siblings of young patients with vascular disease.
Ethnic Variation and In Vivo Effects of the −93t→g Promoter Variant in the Lipoprotein Lipase Gene
Authors: Ehrenborg Ewa , Clee Susanne M. , Pimstone Simon N. , Reymer Paul W.A. , Benlian Pascale , Hoogendijk Christiaan F. , Davis Henry J. , Bissada Nagat , Miao Li , Gagné S. Eric , Greenberg L. Jacquie , Henry Ronald , Henderson Howard , Ordovas José M. , Schaefer Ernst J. , Kastelein Johannes J.P. , Kotze Maritha J. , Hayden Michael R.
Published: June 2011

Abstract
Recently, a (t→g) transition at nucleotide −93 in the lipoprotein lipase (LPL) gene promoter has been observed in Caucasians. Here, we have compared the frequency of the −93g carriers in three distinct populations (Caucasians, South African Blacks, and Chinese). The carrier frequency in the Caucasian population was 1.7% (4/232), which was in contrast to the South African Black population, which had a frequency for this allele of 76.4% (123/161) of the individuals tested. This transition was not observed in the Chinese population under study. Near complete linkage disequilibrium between the −93g and the previously described D9N mutation was observed in the Caucasian population but not in South African Blacks. To further assess the ancestral origins of these DNA changes, DNA haplotyping using a CA repeat 5′ to these substitutions was performed. The −93t allele was associated with only a few specific dinucleotide repeat sizes. In contrast, the −93g allele occurred on chromosomes with many different repeat lengths. The broad distribution of repeats on −93g carrying chromosomes, their high frequency in the South African Black population, and the conservation of the −93g allele among different species may suggest that the −93g allele is the ancestral allele on which a transition to t and the D9N mutations arose. The very high frequency of the −93g allele distinct from the N9 allele in a cohort of Black South Africans allowed us to specifically assess the phenotypic effects of the −93g allele on lipids. Individuals homozygous for the g allele at −93 showed mildly decreased triglycerides compared with individuals homozygous for the t allele (1.14±0.66 mmol/L versus 0.82±0.3;
P
=.04). Thus, the −93g allele in this cohort is associated with low plasma triglyceride levels.
Seasonal Variations of Rheological and Hemostatic Parameters and Acute-Phase Reactants in Young, Healthy Subjects
Authors: Fröhlich M. , Sund M. , Russ S. , Hoffmeister A. , Fischer H. G. , Hombach V. , Koenig W.
Published: June 2011

Abstract
The incidence of cardiovascular diseases is increased in winter months. Recent studies have shown seasonal changes in plasma viscosity, fibrinogen, and factor VII activity with elevated levels during winter. An increase in these factors generates a “hypercoagulable state,” which may lead to a rise in cardiovascular morbidity and mortality. It has been suggested that an increase in upper respiratory infections might be the underlying cause for the raised acute-phase reactants, in particular fibrinogen, during the winter season. We investigated seasonal variations of 26 parameters, determining blood rheology and hemostasis in 16 healthy volunteers (8 men and 8 women) aged 20 to 41 years. They were seen at monthly intervals over a period of 1 year. Seasonal variation with peak fitted values in the winter months was found for plasma viscosity (
P
<.001 for the seasonal difference), red blood cell deformability (
P
<.001), whole blood viscosity (
P
<.001), hemoglobin (
P
<.001), hematocrit (
P
<.001), mean corpuscular volume (
P
=.001), platelet count (
P
=.01), α1-glycoprotein (
P
<.001), fibrinogen (measured by immunonephelometry;
P
<.001), plasminogen activator inhibitor-1 (
P
=.002), LDL cholesterol (
P
=.003), and triglyceride levels (
P
<.001). HDL cholesterol (
P
<.001) and cortisol (
P
=.001) showed inverse seasonal patterns, with a maximum during summertime. No statistically significant seasonal variations were seen for red blood cell aggregation, complement factor C4, total cholesterol, ceruloplasmin, haptoglobin, white blood cell count, and plasminogen. These data do not support the hypothesis that increased morbidity and mortality from cardiovascular diseases during winter may be mainly attributable to increased synthesis of acute-phase proteins due to infections. The cause for the seasonal variations in rheological and hemostatic parameters remains unclear and should be studied in more detail.
Enhanced Endothelin-B-Receptor–Mediated Vasoconstriction of Small Porcine Coronary Arteries in Diet-Induced Hypercholesterolemia
Authors: Hasdai David , Mathew Verghese , Schwartz Robert S. , Smith Leslie A. , Holmes David R. , Katusic Zvonimir S. , Lerman Amir
Published: June 2011

Abstract
The coronary vasoconstrictor effects of endothelins, mediated by both endothelin ETA and ETB receptors, may be differentially altered in pathophysiological states associated with endothelial dysfunction and elevated endothelin levels. Experimental hypercholesterolemia is associated with coronary endothelial dysfunction and increased circulating endothelin concentrations. These studies were designed to test the hypothesis that experimental hypercholesterolemia is characterized by a differentially altered coronary contractile response to ETA- and ETB-receptor stimulation, in vitro. Pigs were fed either a normal or a high-cholesterol diet for 10 to 13 weeks. Changes in the intraluminal diameter of pressurized small coronary arteries (<481±25 μm in diameter) to cumulative concentrations (10
−10
to 10
−6
mol/L) of endothelin-1 (ET-1), and sarafotoxin 6c (S6c), a specific ETB-receptor agonist, were measured using a video dimension analyzer. The maximal contraction attained with ET-1 was greater than with S6c in both normal (86±7% versus 47±7%,
P
=.001) and hypercholesterolemic (77±6% versus 37±7%,
P
<.001) pigs. At 10
−10
mol/L, vessels from hypercholesterolemic pigs manifested greater contraction to both ET-1 (23±6% versus 8±3%,
P
=.02) and S6c (17±5% versus 4±2%,
P
=.02). Incubation of arteries from hypercholesterolemic pigs with BQ-788 (ETB-receptor antagonist), but not FR-139317 (ETA-receptor antagonist), altered the contractile response to ET-1 at 10
−10
mol/L. Removal of the endothelium abolished the difference in response to S6c between normal and hypercholesterolemic pigs. These studies demonstrate that experimental hypercholesterolemia is characterized by enhanced coronary vasoconstriction to endothelins in vitro, the mechanism of which is mediated mainly through the ETB receptor. Thus, the ETB receptor has a role in regulation of coronary artery tone in both the steady-state and pathophysiological states.
Vascular Endothelial Growth Factor/Vascular Permeability Factor Produces Nitric Oxide–Dependent Hypotension
Authors: Horowitz Jeffrey R. , Rivard Alain , van der Zee Rien , Hariawala Mukesh , Sheriff Don D. , Esakof Darryl D. , Chaudhry G. Muqtada , Symes James F. , Isner Jeffrey M.
Published: June 2011

Abstract
In vitro studies suggest that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) may stimulate release of nitric oxide (NO) from endothelial cells. To investigate the hemodynamic consequences of recombinant VEGF/VPF administered in vivo, recombinant human VEGF/VPF was administered as a bolus dose of 500 μg to anesthetized (n=6) or conscious (n=5) New Zealand White rabbits, as well as anesthetized rabbits with diet-induced hypercholesterolemia (HC; n=7). Anesthetized Yorkshire farm pigs (no specific dietary pretreatment) were studied before and after receiving 500 μg intravenous (IV; n=5) or intracoronary (IC; n=5) VEGF/VPF. In anesthetized, normal rabbits, mean arterial pressure (MAP) fell by 20.5±1.4% (
P
<.05 versus baseline) within 3 minutes after IV VEGF/VPF. Pretreatment with
N
ω
-nitro-
l
-arginine caused a significant inhibition of VEGF/VPF-induced hypotension. In conscious, normal rabbits, VEGF/VPF produced a consistent though lesser reduction in MAP. The fall in MAP induced by VEGF/VPF in anesthetized, HC rabbits (21.5±2.5% from baseline) was no different from that observed in normal anesthetized rabbits. In pigs, both IV and IC administration of VEGF/VPF produced a prompt reduction in MAP. Heart rate increased, while cardiac output, stroke volume, left atrial pressure, and total peripheral resistance all declined to a similar, statistically significant degree in both IV and IC groups. Epicardial echocardiography disclosed neither global nor segmental wall motion abnormalities in response to VEGF/VPF. We conclude that (1) VEGF/VPF-stimulated release of NO, previously suggested in vitro, occurs in vivo; (2) this finding suggests that functional VEGF/VPF receptors are present on quiescent adult endothelium, consistent with a maintenance function for VEGF/VPF, which may include regulation of NO; and (3) the preserved response of HC rabbits suggests that endothelial cell receptors for VEGF/VPF are spared in the setting of hypercholesterolemia.
The Arg
353
Gln Polymorphism Reduces the Level of Coagulation Factor VII
Authors: Hunault Mathilde , Arbini Arnaldo A. , Lopaciuk Stanislaw , Carew Josephine A. , Bauer Kenneth A.
Published: June 2011

Abstract
Factor VII levels are regulated by environmental and genetic factors. Two polymorphisms, a G-to-A transversion at nucleotide 10976 resulting in Arg
353
Gln and a decanucleotide insert at position -323 in the 5′-flanking region of the factor VII gene, have been associated with a 20% to 25% reduction in plasma factor VII levels. However Arg
353
Gln almost always segregates on alleles containing the insert in UK and Italian populations, thereby making it impossible to independently evaluate the impact of Arg
353
Gln on factor VII levels in these ethnic groups. We have evaluated the influence of genotype on factor VII levels in 99 healthy Polish blood donors and observed that Arg
353
Gln frequently occurs in the absence of the insert. In univariate analysis, the mean levels of factor VII coagulant activity (VII:C) and factor VII antigen (VII:Ag) were significantly lower in 16 people who were heterozygous for Arg
353
Gln and the insert compared with 72 normal subjects who had neither Arg
353
Gln nor the insert (88.8% of normal and 83.1% versus 102% and 100%,
P
=.019 and
P
=.0003, respectively). In nine subjects heterozygous for Arg
353
Gln alone, VII:C and VII:Ag were significantly decreased compared with the normal subjects (81.9% and 83%, respectively,
P
=.007 and
P
=.004). In multivariate analysis, Arg
353
Gln but not the insert significantly reduced VII:C and VII:Ag after adjustment for age and plasma triglycerides (
P
<.05 and
P
=.02, respectively). To evaluate the mechanism responsible for reduced factor VII levels in individuals with Arg
353
Gln, we performed transient transfection assays with factor VII cDNA containing the base substitution resulting in Gln
353
and wild-type factor VII cDNA in COS-1 cells. The levels of VII:Ag in the cell lysates were similar, but the amino acid substitution significantly reduced factor VII secretion into the media to 74.9% of wild-type (
P
=.0001). Based on these in vivo and in vitro studies, we conclude that the Arg
353
Gln polymorphism alone can decrease plasma factor VII levels.
Induction of Vascular SMC Proliferation by Urokinase Indicates a Novel Mechanism of Action in Vasoproliferative Disorders
Authors: Kanse Sandip M. , Benzakour Omar , Kanthou Chryso , Kost Christine , Lijnen H. Roger , Preissner Klaus T.
Published: June 2011

AbstractThe urokinase-type plasminogen activator (UPA) and its receptor are expressed in the vasculature and are involved in cell migration and remodeling of the extracellular matrix in the neointima. Vessels with atherosclerosis or neointimal hyperplasia, when compared with normal vessels, contain high UPA activity as well as increased levels of UPA receptor. In this study, we have identified the stimulation of vascular smooth muscle cell proliferation as a novel activity for UPA in the vessel wall. High-molecular-weight-UPA (12-200 nmol/L range) stimulated DNA synthesis and cell proliferation, which was half that induced by fetal calf serum or by platelet-derived growth factor-BB. UPA did not induce growth of endothelial cells, and tissue-type plasminogen activator showed no activity on either cell type. Induction of proliferation required the complete UPA molecule but was independent of the proteolytic activity of UPA, whereas neither the amino-terminal fragment nor the catalytic domain by itself was mitogenic. UPA also stimulated c-fos/c-mycmRNA expression and mitogen-activated protein kinase activity in smooth muscle cells. Blocking monoclonal antibodies against the UPA receptor and the enzymatic removal of receptors were ineffective in inhibiting the mitogenic effect of UPA, suggesting a UPA receptor–independent mechanism. Thus, we provide evidence for a novel function of UPA on vascular smooth muscle cell proliferation that, together with its previously documented involvement in regulating pericellular proteolysis-related events and cell migration, provides additional evidence for a role in the pathogenesis of atherosclerosis/restenosis.
Genistein, the Dietary-Derived Angiogenesis Inhibitor, Prevents LDL Oxidation and Protects Endothelial Cells From Damage by Atherogenic LDL
Authors: Kapiotis S. , Hermann M. , Held I. , Seelos C. , Ehringer H. , Gmeiner B. M. K.
Published: June 2011

Abstract
There is now growing evidence that the oxidative modification of LDL plays a potential role in atherosclerosis. In this study, genistein, a compound derived from a soy diet with a flavonoid chemical structure (4′,5,7-trihydroxyisoflavone), which was found to inhibit angiogenesis, has been evaluated for its ability to act as an LDL antioxidant and a vascular cell protective agent against oxidized LDL. The results showed that genistein was able to inhibit the oxidation of LDL in the presence of copper ions or superoxide/nitric oxide radicals as measured by thiobarbituric acid-reactive substance formation, alteration in electrophoretic mobility, and lipid hydroperoxides. Bovine aortic endothelial cell- and human endothelial cell-mediated LDL oxidation was also inhibited in the presence of genistein. The 7-
O
-glucoside of genistein, genistin, was much less effective in inhibiting LDL oxidation in the cell-free and cell-mediated lipoprotein-oxidating systems. Incubating human endothelial cells in the absence or presence of genistein and challenging the cells with already oxidized lipoprotein revealed that in addition to its antioxidative potential during LDL oxidating processes, genistein effectively protected the vascular cells from damage by oxidized lipoproteins. The tyrosine kinase inhibitor genistein was found to block upregulation of two tyrosine-phosphorylated proteins of 132 and 69 kDa in endothelial cells induced by oxidized LDL. Parallel experiments with the inactive analogue daidzein, however, showed that the cytoprotective effect of the isoflavones seems not to be dependent on tyrosine phosphorylation. Our findings will support the suggested and documented beneficial action of a soy diet in preventing chronic vascular diseases and early atherogenic events.
A Prothrombin Gene Mutation Is Significantly Associated With Venous Thrombosis
Authors: Kapur Rajesh K. , Mills Lisa A. , Spitzer Silvia G. , Hultin Mae B.
Published: June 2011

Abstract
This case-control study examined the prevalence of a prothrombin gene mutation in the 3′-untranslated region (UTR) first reported by Poort et al in Dutch subjects with a history of venous thrombosis and in matched control subjects without a history of thrombosis. We tested the hypothesis that the presence of the 3′UTR prothrombin mutation would convey a higher risk of venous or arterial thrombosis and therefore would be found in a higher-than-normal percentage of subjects with a history of thrombosis. Our study included 100 subjects: 50 with a history of thrombosis (21 with venous thrombosis and 29 with arterial thrombosis, who had been recruited from an anticoagulation clinic) and 50 control subjects without a history of thrombosis. DNA from these subjects was analyzed by polymerase chain reaction and agarose gel electrophoresis. We found a statistically significant increase in the prevalence of the 3′UTR mutation in subjects with a history of venous thrombosis compared with subjects without thrombosis. The prevalence of the 3′UTR prothrombin mutation was 19% (4/21; 3 heterozygous and 1 homozygous) in subjects with a history of venous thrombosis, 0% (0/29) in subjects with a history of arterial thrombosis, and 2% (1/50) in control subjects (
P
<.0245, by Fisher’s exact test for comparison of subjects with versus those without a history of venous thrombosis). The G→A mutation at nucleotide 20 210 in the 3′UTR was confirmed by direct DNA sequencing. The similar increased prevalence of the 3′UTR mutation in subjects with venous thrombosis in our population and in the Dutch population studied by Poort et al suggests that this mutation is an important risk factor for venous thrombosis in the general white population.
Suppression of Lipid Transfer Inhibitor Protein Activity by Oleate
Authors: Morton Richard E. , Greene Diane J.
Published: June 2011

Abstract
Cholesteryl ester transfer protein (CETP) mediates the interlipoprotein exchange of cholesteryl ester (CE) and triglyceride. A second plasma protein, lipid transfer inhibitor protein (LTIP), binds to lipoproteins and inhibits CETP activity by displacing CETP from the lipoprotein surface. Since free fatty acids (FFAs) enhance the binding of CETP to lipoproteins, we have examined the possible role of FFAs in modulating LTIP activity. Partially purified CETP, LTIP, and lipoproteins were incubated with 0 to 30 μmol/L sodium oleate, and the transfer of CE between a labeled donor lipoprotein and a given acceptor lipoprotein was measured. Without LTIP, oleate stimulated CETP-mediated CE transfer between VLDL, LDL, and HDL up to threefold. This stimulation was unique in both magnitude and oleate concentration dependence for each donor-acceptor lipoprotein pair. In contrast to CETP activity, in transfer reactions involving LDL or VLDL as donor, LTIP activity was suppressed (>80%) by 10 to 15 μmol/L oleate. LTIP activity in transfer reactions with HDL as donor was less sensitive. Similar results to these were observed when lipid transfer reactions were measured in the total lipoprotein fraction isolated from FFA-enriched plasma. The FFA content of lipoproteins was strongly influenced by the concentration of FFA in plasma; lipoprotein FFA levels sufficient to suppress LTIP activity by 50% to 100% were achieved in plasma containing 0.8 to 1.0 mmol/L FFA. We conclude that LTIP may be functionally inactive during periods of transient elevations of plasma FFA levels, such as during postprandial lipemia or overnight fasting, or chronically suppressed in disease states in which plasma FFA levels are increased. The suppression of LTIP activity by FFA allows for maximum CETP-mediated lipid transfer between all lipoproteins, including lipid transfer reactions involving LDL that are normally preferentially suppressed by LTIP.
Human Monocyte–Derived Macrophages Express an ≈120-kD Ox-LDL Binding Protein With Strong Identity to CD68
Authors: van der Kooij Maaike A. , von der Mark Elisabeth M. , Kruijt J. Kar , van Velzen Agnes , van Berkel Theo J.C. , Morand Olivier H.
Published: June 2011

AbstractA protein that specifically binds oxidized LDL (Ox-LDL) has recently been characterized in mouse peritoneal macrophages and identified as macrosialin, a protein with a molecular weight of 95 kD. First, the present work shows that human monocyte–derived macrophages express a membrane protein with a molecular weight of ≈120 kD that selectively binds Ox-LDL. Second, we tested whether this ≈120-kD Ox-LDL binding protein had any relation to CD68, the human homologue of macrosialin. The following evidence was obtained to support the role of CD68 as an Ox-LDL binding protein: (1) Ligand blots with Ox-LDL and Western blots with Ki-M6, an anti–human CD68 monoclonal antibody, revealed a single band with a molecular weight of ≈120 kD under reducing and nonreducing condition. (2) The expression patterns of the ≈120-kD Ox-LDL binding membrane protein and of CD68 paralleled each other during monocyte/macrophage differentiation. (3) Digestion withN-glycosidase F demonstrated that both CD68 and the Ox-LDL binding protein are glycoproteins; both showed a similar shift of ≈18 kD in apparent molecular weight. (4) CD68, probed with monoclonal antibody Ki-M6, and the ≈120-kD Ox-LDL binding protein were coprecipitated with EBM11, another anti-CD68 antibody. About 5000 molecules of CD68 are expressed on the cell surface of human macrophages. Ligation of125I–Ki-M6 to cells leads to its internalization and degradation. This capacity would be sufficient to allow for the specific uptake and degradation of Ox-LDL. Taken together, these data support a role for CD68 as a specific Ox-LDL binding protein in human monocyte–derived macrophages.