bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation and Characterization of Human Smooth Muscle Cell Lines Derived From Atherosclerotic Plaque
Authors: Bonin Lisé R. , Madden Karen , Shera Katherine , Ihle Jennifer , Matthews Connie , Aziz Salim , Perez-Reyes Nuria , McDougall James K. , Conroy Stephen C.
Published: June 2011

Abstract
—The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly −Y, +7, −13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2 novel plaque cell lines exhibiting various features of plaque SMC biology; pdSMC2 may represent an earlier plaque SMC phenotype, whereas pdSMC1A may be representative of cells comprising an advanced atherosclerotic lesion.
Microalbuminuria and Peripheral Arterial Disease Are Independent Predictors of Cardiovascular and All-Cause Mortality, Especially Among Hypertensive Subjects
Authors: Jager Agnes , Kostense Piet J. , Ruhé Henricus G. , Heine Robert J. , Nijpels Giel , Dekker Jacqueline M. , Bouter Lex M. , Stehouwer Coen D. A.
Published: June 2011

Abstract
—Microalbuminuria (MA) is associated with increased cardiovascular and all-cause mortality. It has been proposed that MA reflects generalized atherosclerosis and may thus predict mortality. To investigate this hypothesis, we studied the associations between, on the one hand, MA and peripheral arterial disease (PAD), a generally accepted marker of generalized atherosclerosis, and, on the other hand, cardiovascular and all-cause mortality in an age-, sex-, and glucose tolerance-stratified sample (n=631) of a population-based cohort aged 50 to 75 years followed prospectively for 5 years. At baseline, the albumin-to-creatinine ratio (ACR) was measured in an overnight spot urine sample; MA was defined as ACR >2.0 mg/mmol. PAD was defined as an ankle-brachial pressure index below 0.90 and/or a history of a peripheral arterial bypass or amputation. After 5 years of follow-up, 58 subjects had died (24 of cardiovascular causes). Both MA and PAD were associated with a 4-fold increase in cardiovascular mortality. After adjusting for age, sex, diabetes mellitus, hypertension, levels of total and HDL-cholesterol and triglyceride, body mass index, smoking habits, and preexistent ischemic heart disease, the relative risks (RR) (95% confidence intervals) were 3.2 (1.3 to 8.1) for MA and 2.4 (0.9 to 6.1) for PAD. When both MA and PAD were included in the multivariate analysis, the RRs were 2.9 (1.1 to 7.3) for MA and 2.0 (0.7 to 5.7) for PAD. MA and PAD were both associated with an about 2-fold increase in all-cause mortality. The RRs of all-cause mortality associated with MA and PAD were about 4 times higher among hypertensive than among normotensive subjects. We conclude that both MA and PAD are associated with an increased risk of cardiovascular mortality. MA and PAD are mutually independent risk indicators. The associations of MA and PAD with all-cause mortality are somewhat weaker. They are more pronounced in the presence of hypertension than in its absence. These data suggest that MA affects mortality risk through a mechanism different from generalized atherosclerosis.
Interaction Between the G20210A Mutation of the Prothrombin Gene and Oral Contraceptive Use in Deep Vein Thrombosis
Authors: Martinelli Ida , Taioli Emanuela , Bucciarelli Paolo , Akhavan Sepideh , Mannucci Pier Mannuccio
Published: June 2011

Abstract
—Single-point mutations in the gene coding for prothrombin (factor II:A20210) or factor V (factor V:A1691) are associated with an increased risk of venous thromboembolism. The use of oral contraceptives is also a strong and independent risk factor for the disease, and the interaction between factor V:A1691 and oral contraceptives greatly increases the risk. No information is available about the interaction between oral contraceptives and mutant prothrombin. We investigated 148 women with a first, objectively confirmed episode of deep vein thrombosis and 277 healthy women as controls. Fourteen patients (9.4%) were carriers of factor II:A20210, 24 (16.2%) of factor V:A1691, and 4 (2.7%) of both defects. Among controls, the prevalence was 2.5% for either factor II:A20210 or factor V:A1691, and there was no carrier of both the mutations. The relative risk of thrombosis was 6-fold for factor II:A20210 and 9-fold for factor V:A1691. The most prevalent circumstantial risk factor in patients and the only one observed in controls was oral contraceptive use, which per se conferred a 6-fold increased risk of thrombosis. The risk increased to 16.3 and 20.0 when women with factor II:A20210 or factor V:A1691 who used oral contraceptives were compared with noncarriers and nonusers. These figures indicate a multiplicative interaction between the genetic risk factors and oral contraceptives. No difference in the type of oral contraceptives was observed between patients and controls, those of third generation being the most frequently used (73% and 80%). We conclude that carriers of the prothrombin mutation who use oral contraceptives have a markedly increased risk of deep vein thrombosis, much higher than the risk conferred by either factor alone.
Menopausal Status and Distensibility of the Common Carotid Artery
Authors: Westendorp Iris C. D. , Bots Michiel L. , Grobbee Diederick E. , Reneman Robert S. , Hoeks Arnold P. G. , Van Popele Nicole M. , Hofman Albert , Witteman Jacqueline C. M.
Published: June 2011

Abstract
—Although several studies have shown that exogenous estrogens have beneficial effects on arterial characteristics, the effect of endogenous estrogen on the vascular system is still unknown. In this study, distensibility, an indicator of arterial elasticity, of the common carotid artery was compared in pre- and postmenopausal women. The study comprised 93 premenopausal and 93 postmenopausal women of similar age (range, 43 to 55 years). Women were selected from respondents to a mailed questionnaire about the menopause, which was sent to all women aged 40 to 60 years in the Dutch town of Zoetermeer (n=12 675). Postmenopausal women who were at least 3 years past natural menopause or whose menses had stopped naturally before age 48, were age-matched with premenopausal women with regular menses and without menopausal complaints. The selection aimed at maximizing the contrast in estrogen status between pre- and postmenopausal women of the same age. Distensibility of the carotid artery was measured noninvasively with B-mode ultrasound and a vessel wall movement detector system. Arterial distensibility is expressed as the change in arterial diameter (distension, ΔD) with the cardiac cycle, adjusted for lumen diameter, pulse pressure, and mean arterial blood pressure. Compared with premenopausal women, postmenopausal women had significantly lower arterial distension (ΔD 370.5 μm [SE 9.5] versus 397.3 μm [SE 9.6]). These results suggest that the distensibility of the common carotid artery is negatively affected by natural menopause in presumed healthy women.
Formation of Hyaluronan- and Versican-Rich Pericellular Matrix Is Required for Proliferation and Migration of Vascular Smooth Muscle Cells
Authors: Evanko Stephen P. , Angello John C. , Wight Thomas N.
Published: June 2011

Abstract
—The accumulation of hyaluronan (HA) and the HA-binding proteoglycan versican around smooth muscle cells in lesions of atherosclerosis suggests that together these molecules play an important role in the events of atherogenesis. In this study we have examined the formation of HA- and versican-rich pericellular matrices by human aortic smooth muscle cells in vitro, using a particle-exclusion assay, and the role of the pericellular matrix in cell proliferation and migration. The structural dependence of the pericellular matrix on HA can be demonstrated by the complete removal of the matrix with
Streptomyces
hyaluronidase. The presence of versican in the pericellular matrix was confirmed immunocytochemically. By electron microscopy, the cell coat was seen as a tangled network of hyaluronidase-sensitive filaments decorated with ruthenium red–positive proteoglycan granules. Ninety percent of migrating cells in wounded cultures, and virtually all mitotic cells, displayed abundant HA- and versican-rich coats. Time-lapse video imaging revealed that HA- and versican-rich pericellular matrix formation is dynamic and rapid, and coordinated specifically with cell detachment and mitotic cell rounding. HA oligosaccharides, which inhibit the binding of HA to the cell surface and prevent pericellular matrix formation, significantly reduced proliferation and migration in response to platelet-derived growth factor, whereas larger HA fragments and high molecular weight HA had no effect. Treatment with HA oligosaccharides also led to changes in cell shape from a typical fusiform morphology to a more spread and flattened appearance. These data suggest that organization of HA- and versican-rich pericellular matrices may facilitate migration and mitosis by diminishing cell surface adhesivity and affecting cell shape through steric exclusion and the viscous properties of HA proteoglycan gels.
Poor Glycemic Control Predicts Coronary Heart Disease Events in Patients With Type 1 Diabetes Without Nephropathy
Authors: Lehto Seppo , Rönnemaa Tapani , Pyörälä Kalevi , Laakso Markku
Published: June 2011

Abstract
—Patients with type 1 diabetes mellitus, especially those with nephropathy, are at increased risk for coronary heart disease (CHD). However, information on the predictive value of cardiovascular risk factors and the degree of hyperglycemia with respect to CHD events in patients with type 1 diabetes without nephropathy is still incomplete. Therefore, we performed a prospective study on risk factors for CHD in patients with type 1 diabetes free of clinical nephropathy. At baseline examination, cardiovascular risk factor levels of CHD were determined in 177 patients with type 1 diabetes (87 men and 90 women), age 45 to 64 years at baseline and ≥30 years at the time of diagnosis of diabetes. These patients were followed up to 7 years with respect to CHD events. Altogether, 20 patients with type 1 diabetes (13 men [7.3%] and 7 women [3.9%]) died of CHD and 28 patients with type 1 diabetes (17 men [9.6%] and 11 women [6.2%]) had a serious CHD event (death from CHD or nonfatal myocardial infarction). In multivariate Cox regression analysis, a previous history of myocardial infarction (hazard ratio [HR] and its 95% confidence interval, 8.0 [3.1 to 21.0],
P
<0.001), high glycohemoglobin A
1
(>10.4%, the highest tertile, HR 5.4 [1.4 to 20.4],
P
=0.013), and the duration of diabetes (>16 years, the highest tertile, HR 4.2 [1.3 to 12.9],
P
=0.013) were the only variables associated with CHD death even after adjustment for other cardiovascular risk factors. These variables also predicted the incidence of all CHD events. Our results indicate that poor metabolic control is a strong predictor of CHD events in patients with late-onset type 1 diabetes without nephropathy, independently of other cardiovascular risk factors.
Coagulation Factors II, V, VII, and X, Prothrombin Gene 20210G→A Transition, and Factor V Leiden in Coronary Artery Disease
Authors: Redondo M. , Watzke H. H. , Stucki B. , Sulzer I. , Biasiutti F. Demarmels , Binder B. R. , Furlan M. , Lämmle B. , Wuillemin W. A.
Published: June 2011

Abstract
—Increased levels of hemostatic factors and genetic mutations of proteins involved in coagulation may play a role in the pathogenesis of coronary artery disease. We investigated clotting activity of factors II (FII:C), V (FV:C), VII (FVII:C), and X (FX:C), the prothrombin gene 20210G→A transition, and the factor V Leiden mutation in 200 survivors of myocardial infarction and in 100 healthy controls. FV:C (
P
<0.0001) and FVII:C (
P
<0.0001) were found to be independent risk factors for myocardial infarction. High FV:C or high FVII:C combined with smoking or arterial hypertension increased the relative risk for myocardial infarction up to 50-fold. One of 177 patients (0.6%) and 4 of 89 controls (4.5%) had the prothrombin 20210 AG genotype. Eleven of 177 patients (6.2%) and 6 of 89 controls (6.7%) were heterozygous for the factor V Leiden mutation. No homozygous carrier for these mutations was found. Neither the prothrombin gene 20210G→A transition (odds ratio [OR], 0.1; 95% confidence interval [CI], 0.01 to 1.1) nor the factor V Leiden mutation (OR, 1.0; 95% CI, 0.4 to 2.8) were associated with an increased relative risk for myocardial infarction. In conclusion, our data indicate that neither the prothrombin gene 20210G→A transition nor the factor V Leiden mutation are risk factors for myocardial infarction. High FVII:C was confirmed to be an independent risk factor for myocardial infarction. Moreover, we describe for the first time that high FV:C is an independent risk factor for myocardial infarction.
Increased Platelet Aggregability Associated With Platelet

GPIIIa Pl

A2

Polymorphism
Authors: Feng DaLi , Lindpaintner Klaus , Larson Martin G. , Rao Valluri S. , O’Donnell Christopher J. , Lipinska Izabella , Schmitz Christian , Sutherland Patrice A. , Silbershatz Halit , D’Agostino Ralph B. , Muller James E. , Myers Richard H. , Levy Daniel , Tofler Geoffrey H.
Published: June 2011

Abstract
—The platelet glycoprotein
IIb/IIIa (GP IIb/IIIa)
plays a pivotal role in platelet aggregation. Recent data suggest that the
Pl

A2

polymorphism of
GPIIIa
may be associated with an increased risk for cardiovascular disease. However, it is unknown if there is any association between this polymorphism and platelet reactivity. We determined
GP IIIa
genotype and platelet reactivity phenotype data in 1422 subjects from the Framingham Offspring Study. Genotyping was performed using PCR-based restriction fragment length polymorphism analysis. Platelet aggregability was evaluated by the Born method. The threshold concentrations of epinephrine and ADP were determined. Allele frequencies of
Pl

A1

and
Pl

A2

were 0.84 and 0.16, respectively. The presence of 1 or 2
Pl

A2

alleles was associated with increased platelet aggregability as indicated by incrementally lower threshold concentrations for epinephrine and ADP. For epinephrine, the mean concentrations were 0.9 μmol/L (0.9 to 1.0) for homozygous
Pl

A1

, 0.7 mmol/L (0.7 to 0.9) for the heterozygous
Pl

A1

/
Pl

A2

, and 0.6 μmol/L (0.4 to 1.0) for homozygous
Pl

A2

individuals,
P
=0.009. The increase in aggregability induced by epinephrine remained highly significant (
P
=0.007) after adjustment for covariates. For ADP-induced aggregation, the respective mean concentrations were 3.1 μmol/L (3.0 to 3.2), 3.0 μmol/L (2.9 to 3.2), and 2.8 μmol/L (2.4 to 3.3);
P
=0.19 after adjustment for covariates. Our findings indicate that molecular variants of the gene encoding
GP IIIa
play a role in platelet reactivity in vitro. Our observations are compatible with and provide an explanation for the reported association of the
Pl

A2

allotype with increased risk for cardiovascular disease.
Evidence of Hypoxic Areas Within the Arterial Wall In Vivo
Authors: Björnheden T. , Levin M. , Evaldsson M. , Wiklund O.
Published: June 2011

Abstract
—The anoxemia theory of atherosclerosis states that an imbalance between the demand and supply of oxygen in the arterial wall is a key factor for the development of atherosclerotic lesions. Direct in vitro and in situ measurements have shown that P
o
2
is decreased in the more deeply situated parts of the media, but the degree of hypoxia in vivo or the distribution of hypoxia along the arterial tree is not known. For this reason, we have developed a method for the detection of hypoxia in the arterial wall in vivo by using a hypoxia marker, 7-(4′-(2-nitroimidazol-1-yl)-butyl)-theophylline, that may be visualized by immunofluorescence. In the present study, we have used this method in rabbits with experimentally induced atherosclerosis. Our results indicate that zones of hypoxia occur at depth in the atherosclerotic plaque. The mechanism was probably an impaired oxygen diffusion capacity due to the thickness of the lesion, together with high oxygen consumption by the foam cells. Thus, we have for the first time demonstrated that hypoxia actually does exist in the arterial wall in vivo, lending support to the anoxemia theory of atherosclerosis.
The Role of Alpha and Beta Platelet-Derived Growth Factor Receptor in the Vascular Response to Injury in Nonhuman Primates
Authors: Giese Neill A. , Marijianowski Monique M. H. , McCook Oscar , Hancock Andrea , Ramakrishnan Vanitha , Fretto Larry J. , Chen Changyi , Kelly Andrew B. , Koziol James A. , Wilcox Josiah N. , Hanson Stephen R.
Published: June 2011

Abstract
—Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (
P
<0.05) and 48% (
P
<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis.
Uptake of 13-Hydroperoxylinoleic Acid by Cultured Cells
Authors: Augé Nathalie , Santanam Nalini , Mori Natsuko , Keshava Channa , Parthasarathy Sampath
Published: June 2011

Abstract—Oxidized free fatty acids have profound effects on cultured cells. However, little is known about whether these effects depend on their uptake and metabolism by cells or primarily involve their interaction with cell-surface components. We determined the uptake and metabolism of unoxidized (linoleic or oleic acid) and oxidized linoleic acid (13-hydroperoxyoctadecadienoic acid, 13-HPODE) by endothelial cells, smooth muscle cells, and macrophages. We show that 13-HPODE is poorly taken up by cells. The levels of uptake were dependent on the cell type but were independent of the expression of CD36. 13-HPODE was also poorly used by microsomal lysophosphatidylcholine acyltransferase that is involved in the formation of phosphatidylcholine. Based on these results, we suggest that most of the biological effects of 13-HPODE and other oxidized free fatty acids on cells might involve a direct interaction with cell-surface components. Alternatively, very small amounts of oxidized free fatty acids that enter the cell may have effects, analogous to those of hormones or prostanoids.
A New Promoter Polymorphism in the Gene of Lipopolysaccharide Receptor CD14 Is Associated With Expired Myocardial Infarction in Patients With Low Atherosclerotic Risk Profile
Authors: Unkelbach Katja , Gardemann Andreas , Kostrzewa Markus , Philipp Monika , Tillmanns Harald , Haberbosch Werner
Published: June 2011

Abstract—Recent findings suggest that inflammation plays a role in atherosclerosis and its acute complications. Cellular response in infections with Gram-negative bacteria is mediated by bacterial lipopolysaccharide (LPS), which activates monocytes to expression of cytokines, growth factors, and procoagulatory factors via LPS receptor CD14. Endothelial cells and smooth muscle cells are stimulated by a complex of LPS and soluble CD14. In this study, LPS receptor CD14 was analyzed to find genetic variants and check them for an association with coronary artery disease or myocardial infarction (MI). When screening the CD14 gene by single-strand conformation polymorphism analysis, a promoter polymorphism was detected and confirmed as a T-to-C exchange at position −159. We determined the genotypes of 2228 men who had undergone coronary angiography for diagnostic purposes. Within the total study group there was no significant association of either genotype with MI or coronary artery disease. However, in a subgroup with low coronary risk (normotensive nonsmokers), a relative risk for MI in probands homozygous for the T allele could be evaluated (OR, 1.6; 95% CI, 1.0 to 2.4;P<0.05). The association was even stronger in low-risk patients older than 62 years (OR, 3.8; 95% CI, 1.6 to 9.0;P<0.01). In conclusion, we describe a new CD14 promoter polymorphism that is associated with MI, especially in older patients with a low atherosclerotic risk profile.
ApoB100 Secretion From HepG2 Cells is Decreased by the ACAT Inhibitor CI-1011
Authors: Wilcox Lisa J. , Barrett P. Hugh R. , Newton Roger S. , Huff Murray W.
Published: June 2011

Abstract
—The concept that hepatic cholesteryl ester (CE) mass and the rate of cholesterol esterification regulate hepatocyte assembly and secretion of apoB-containing lipoproteins remains controversial. The present study was carried out in HepG2 cells to correlate the rate of cholesterol esterification and CE mass with apoB secretion by CI-1011, an acyl CoA:cholesterol acyltransferase (ACAT) inhibitor that is known to decrease apoB secretion, in vivo, in miniature pigs. HepG2 cells were incubated with CI-1011 (10 nmol/L, 1 μmol/L, and 10 μmol/L) for 24 hours. ApoB secretion into media was decreased by 25%, 27%, and 43%, respectively (
P
<0.0012). CI-1011 (10 μmol/L) inhibited HepG2 cell ACAT activity by 79% (
P
<0.002) and cellular CE mass by 32% (
P
<0.05). In contrast, another ACAT inhibitor, DuP 128 (10 μmol/L), decreased cellular ACAT activity and CE mass by 85% (
P
<0.002) and 42% (
P
=0.01), respectively, but had no effect on apoB secretion into media. To characterize the reduction in apoB secretion by CI-1011, pulse-chase experiments were performed and analyzed by multicompartmental modelling using SAAM II. CI-1011 did not affect the synthesis of apoB or albumin. However, apoB secretion into the media was decreased by 42% (
P
=0.019). Intracellular apoB degradation increased proportionately (
P
=0.019). The secretion of albumin and cellular reuptake of labeled lipoproteins were unchanged. CI-1011 and DuP 128 did not affect apoB mRNA concentrations. These results show that CI-1011 decreases apoB secretion by a mechanism that involves an enhanced intracellular degradation of apoB. This study demonstrates that ACAT inhibitors can exert differential effects on apoB secretion from HepG2 cells that do not reflect their efficacy in inhibiting cholesterol esterification.
A Bioluminescence Method for the Mapping of Local ATP Concentrations Within the Arterial Wall, With Potential to Assess the In Vivo Situation
Authors: Levin Max , Björnheden Tom , Evaldsson Margareta , Walenta Stefan , Wiklund Olov
Published: June 2011

Abstract
—According to the anoxemia theory of atherosclerosis, an imbalance between the demand for and supply of oxygen and nutrients in the arterial wall is a key factor in atherogenesis. However, the energy metabolic state of the arterial tissue in vivo is largely unknown. We applied a bioluminescence method, metabolic imaging, to study local ATP concentrations in cryosections of normal pig and atherosclerotic and normal rabbit aorta. Some vessels were subjected to energy metabolic restrictions by incubation at different oxygen and glucose concentrations and others were rapidly frozen in liquid nitrogen to reflect the in vivo situation. Local ATP concentrations and the ATP distribution at a microscale was dependent on oxygen as well as glucose concentrations during incubation. ATP depletion was seen in the mid media of pig aorta in all incubations, but only at low oxygen concentration without glucose in the media of the thinner rabbit aorta. ATP-depleted zones were seen deep in pig media (>750 μm from the lumen) and in rabbit plaques (>300 μm from the lumen) even at high oxygen (pig 75% O
2
and rabbit 21% O
2
) and glucose concentrations (5.6 mmol/L glucose). This observation probably illustrates an insufficient diffusion of glucose, which highlights the importance of studying the conditions for diffusion not only of oxygen but also of other metabolites in the arterial wall. In rapidly frozen vessels the medial ATP concentration was shown to be 0.6 to 0.8 μmol/g wet weight (both pig and rabbit aorta) and in pig aorta a gradient could be seen indicating higher ATP concentrations at the lumenal side. We propose that metabolic imaging, as applied to snap-frozen tissue, may be used to assess the energy metabolic situation in the arterial wall in vivo. The spatial resolution allows the detection of local variations within the arterial tree. However, steep concentration gradients (eg, near the border of the tissue) will be underestimated. The method may be extended to include determinations of glucose and lactate concentrations and will be used in parallel with an established method to assess hypoxia in the arterial wall in vivo.
Farnesol Blocks the L-Type Ca
2+
Channel by Targeting the α
1C
Subunit
Authors: Luft Ulrich C. , Bychkov Rostislav , Gollasch Maik , Gross Volkmar , Roullet Jean-Baptiste , McCarron David A. , Ried Christian , Hofmann Franz , Yagil Yoram , Yagil Chana , Haller Hermann , Luft Friedrich C.
Published: June 2011

Abstract
—We recently demonstrated that farnesol, a 15-carbon isoprenoid, blocks L-type Ca
2+
channels in vascular smooth muscle cells. To elucidate farnesol′s mechanism of action, we performed whole-cell and perforated-patch clamp experiments in rat aortic A7r5 cells and in Chinese hamster ovary (CHO) C9 cells expressing smooth muscle Ca
2+
channel α
1C
subunits. Farnesol dose-dependently and voltage-independently inhibited Ba
2+
currents in both A7r5 and CHOC9 cells, with similar half-maximal inhibitions at 2.6 and 4.3 mmol/L, respectively (
P
=NS). In both cell lines, current inhibition by farnesol was prominent over the whole voltage range without changes in the current-voltage relationship peaks. Neither intracellular infusion of the stable GDP analogue guanosine-5′-
O
-(2-thiodiphosphate) (100 mmol/L) via the patch pipette nor strong conditioning membrane depolarization prevented the inhibitory effect of farnesol, which indicates G protein–independent inhibition of Ca
2+
channels. In an analysis of the steady-state inactivation curve for voltage dependence, farnesol induced a significant, negative shift (≈10 mV) of the potential causing 50% channel inactivation in both cell lines (
P
<0.001). In contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unlike pharmacological Ca
2+
channel blockers, farnesol blocked Ca
2+
currents in the resting state: initial block was 63±8% in A7r5 cells and 50±9% in CHOC9 cells at a holding potential of −80 mV. We then gave 500 mg/kg body weight farnesol by gavage to Sabra hypertensive and normotensive rats and found that farnesol reduced blood pressure significantly in the hypertensive strain for at least 48 hours. We conclude that farnesol may represent an endogenous smooth muscle L-type Ca
2+
channel antagonist. Because farnesol is active in cells expressing only the pore-forming α
1
subunit, the data further suggest that this subunit represents the molecular target for farnesol binding and principal action. Finally, farnesol has a blood pressure–lowering action that may be relevant in vivo.
Role of Matrix Metalloproteinases and Their Tissue Inhibitors in the Regulation of Coronary Cell Migration
Authors: Shi Yi , Patel Sachin , Niculescu Rodica , Chung Wooksung , Desrochers Paul , Zalewski Andrew
Published: June 2011

Abstract
—The migration of vascular cells is regulated by matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Because the activation of adventitial fibroblasts has been implicated in coronary repair, we have examined regional differences in cell outgrowth and the synthesis of MMPs/TIMPs in different layers of porcine coronary arteries. Coronary medial explants demonstrated significantly slower cell outgrowth than coronary adventitia in culture (
P
<0.001). These observations were paralleled by the predominant expression of TIMP-1 and -2 in the media (14-fold and 37-fold higher than in adventitia, respectively,
P
<0.001), whereas higher gelatinolytic activities (MMP-2 and -9) were released from adventitial explants. Smooth muscle cell outgrowth from the media was regulated by endogenous TIMPs, since TIMP inhibition (recombinant MMP-2 or neutralizing anti-TIMP antibodies) facilitated cell outgrowth (
P
<0.001). In contrast, the addition of recombinant TIMP-1 or -2 decreased adventitial cell outgrowth. In the coculture experiments, the presence of coronary media retarded adventitial cell outgrowth, whereas medial damage abrogated these effects, allowing for fibroblast migration (
P
<0.001). In conclusion, this study demonstrated differential migratory properties and distinct MMP/TIMP synthesis by coronary fibroblasts and smooth muscle cells. Endogenous TIMPs in the media may play an important role in maintaining coronary arterial wall homeostasis, whereas high levels of matrix-degrading activities confer the “invasive” characteristics of adventitial fibroblasts.
Proteoglycans Contribution to Association of Lp(a) and LDL With Smooth Muscle Cell Extracellular Matrix
Authors: Lundstam Ulf , Hurt-Camejo Eva , Olsson Gun , Sartipy Peter , Camejo Germán , Wiklund Olov
Published: June 2011

Abstract
—Lp(a) interference with fibrinolysis could contribute to atherothrombosis. Additionally, accumulation of Lp(a) and LDLs, could lead to cholesterol deposition and foam cell formation in atherogenesis. The interactions between Lp(a) and LDL could cause their entrapment in the extracellular matrix of lesions. We found that association of Lp(a) with matrix secreted by cultured human arterial smooth muscle cells increased 2 to 3 times the subsequent specific binding of radioactive LDL. Chondroitin sulfate proteoglycans seem responsible for formation of the specific matrix-Lp(a) and matrix-LDL aggregates. The proteoglycans appeared also to participate in a cooperative increase of radioactive LDL binding to matrix pretreated with Lp(a). In the matrix preincubated with LDL, ≈50% of the additional lipoprotein was bound by ionic interactions. In the matrix preincubated with Lp(a), 20% of the additional LDL was held by ionic bonds, and the rest was held by strong nonionic associations. Binding analysis in physiological solutions confirmed that chondroitin sulfate-rich proteoglycans from the smooth muscle cell matrix have a high affinity for Lp(a) and LDL. The results provide an explanation to the observed localization of Lp(a) and LDL in the extracellular matrix of arterial lesions and suggest a mechanism for their cooperative accumulation there.
Characterization and Comparison of the Mode of Cell Death, Apoptosis Versus Necrosis, Induced by 7β-Hydroxycholesterol and 7-Ketocholesterol in the Cells of the Vascular Wall
Authors: Lizard Gérard , Monier Serge , Cordelet Catherine , Gesquière Laurence , Deckert Valérie , Gueldry Serge , Lagrost Laurent , Gambert Philippe
Published: June 2011

Abstract—Oxidized low density lipoproteins (LDLs) play a central role in atherosclerosis, and their toxicity is due, at least in part, to the formation of oxysterols that have been shown to induce apoptosis in various cell types. As 7β-hydroxycholesterol and 7-ketocholesterol are the major oxysterols found in oxidized LDLs, we have investigated and compared the mode of cell death, apoptosis versus necrosis, that they induce in the cells of the vascular wall, ie, endothelial cells, smooth muscle cells, and fibroblasts. To this end, human vascular endothelial cells from umbilical cord veins (HUVECs), human artery smooth muscle cells, A7R5 rat smooth muscle cells, MRC5 human fibroblasts, and human fibroblasts isolated from umbilical cord veins were taken at confluence and incubated for 48 hours with 7β-hydroxycholesterol or 7-ketocholesterol (concentration range, 5 to 80 μg/mL). In all cells, both 7β-hydroxycholesterol and 7-ketocholesterol exhibited toxic effects characterized by a loss of cell adhesion and an increased permeability to propidium iodide. In oxysterol-treated endothelial and smooth muscle cells, typical features of apoptosis were revealed: condensed and/or fragmented nuclei were detected by fluorescence microscopy after staining with Hoechst 33342, oligonucleosomal DNA fragments were visualized in situ in the cell nuclei by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method, and internucleosomal DNA fragmentation was found on agarose gel. In contrast, in oxysterol-treated fibroblasts, fragmented and/or condensed nuclei were never revealed, and no DNA fragmentation was observed either by the TUNEL method or by DNA analysis on agarose gel, indicating that these oxysterols induced necrosis in these cells but not apoptosis. In addition, acetylated Asp-Glu-Val-l-aspartic acid aldehyde (an inhibitor of Asp-Glu-Val-l-aspartic acid–sensitive caspases) prevented 7β-hydroxycholesterol– and 7-ketocholesterol–induced cell death in HUVECs and smooth muscle cells but not in fibroblasts. Thus, 7β-hydroxycholesterol and 7-ketocholesterol have dual cytotoxic effects on the cells of the vascular wall by their ability to induce apoptosis in endothelial and smooth muscle cells and necrosis in fibroblasts.
Expression of Type VIII Collagen After Cholesterol Diet and Injury in the Rabbit Model of Atherosclerosis
Authors: Plenz Gabriele , Dorszewski Anja , Breithardt G. , Robenek H.
Published: June 2011

Abstract
—This study presents an analysis of the expression of type VIII collagen mRNA in response to cholesterol diet and balloon injury in the rabbit iliac artery. The design of the animal experiments was as follows: 28 male New Zealand White rabbits were divided into the 3 different treatment groups. Group 1 received regular chow; group 2 was fed with a 1% cholesterol diet for 6 weeks and normal chow for 5 weeks; and group 3 underwent balloon injury, then 6 weeks of a 1% cholesterol diet, which was followed by 5 weeks of normal chow. The expression pattern of type VIII collagen mRNA was compared with that of the fibrillar collagen types I and III, transforming growth factor-β1, a factor known to exert the most potent stimulatory effect on collagen synthesis in vitro, and matrix metalloproteinase 1, a collagen-degrading enzyme. The cholesterol diet resulted in an upregulation of type VIII collagen, fibrillar collagens, transforming growth factor-β1, and matrix metalloproteinase I in the adventitia. Although the number of type VIII collagen mRNA–expressing cells in the media increased, no significant difference in overall expression levels was detectable by northern blot analysis. The ratio of medial smooth muscle cells expressing type VIII collagen mRNA to those expressing type I and type III collagen mRNA (CVIII:CI:CIII) changed from 1:1.88:0.03 in the normal media to 1:0.78:0.29. When cholesterol feeding was preceded by balloon injury, type VIII collagen mRNA expression concomitant with the fibrillar collagens was further upregulated over and above that level reported after cholesterol diet alone. In general, low levels of transforming growth factor-β1 mRNA correlated with high expression of matrix metalloproteinase I. Our study indicates that a cholesterol diet resulted in a balanced reorganization of the collagen composition but did not result in marked collagen accumulation. This may provide an extracellular environment that favors migration and proliferation processes during early atherogenesis. It also demonstrates that type VIII collagen is highly expressed and deposited at later stages, and this may be linked to processes such as tissue reorganization during vascular repair and plaque stabilization.
Presence of Oxidized Low Density Lipoprotein in Nonrheumatic Stenotic Aortic Valves
Authors: Olsson Margareta , Thyberg Johan , Nilsson Jan
Published: June 2011

Abstract—The aim of the present study was to analyze if LDL particles trapped in stenotic aortic valve tissue undergo oxidative modification. Degenerative aortic stenosis affects >3% of the population >75 years of age in the Western world. Recent studies have revealed the presence of a chronic inflammatory process similar to what has been described in other degenerative diseases such as atherosclerosis. However, the underlying disease mechanisms of degenerative aortic stenosis still remain largely unknown. Six tricuspid stenotic valves, obtained at valve replacement, were compared with 3 control valves collected from hearts taken out during transplantation. The stenotic valves and the control valves were examined by immunohistochemistry, using antibodies against apoB, 4-hydroxynonenal-modified LDL, leukocytes, and HLA-DR. All valves were also stained with oil red O for neutral lipids. Extracellular neutral lipids were found in all stenotic valves, extending from the bases along the fibrosa layer. This lipid colocalized with apoB- and 4-hydroxynonenal–modified LDL immunoreactivity. 4-Hydroxynonenal–modified LDLs were present around calcium deposits, subendothelially, and in the deeper layer of the fibrosa. There was also a colocalization with macrophages, T lymphocytes, and HLA-DR expression. Control valves had a thin area of neutral lipid accumulation, a small amount of apoB, but no signs of inflammation. A distinct colocalization between oxidized LDLs, T-lymphocyte accumulation, and calcium deposits suggests that oxidized lipids may play a role in the disease process.
Increased Cholesterol Efflux Potential of Sera From ApoA-I
Milano
Carriers and Transgenic Mice
Authors: Franceschini Guido , Calabresi Laura , Chiesa Giulia , Parolini Cinzia , Sirtori Cesare R. , Canavesi Monica , Bernini Franco
Published: June 2011

Abstract
—The ability of HDL to remove cholesterol from peripheral cells and drive it to the liver for excretion is believed to explain most of the strong inverse correlation between plasma HDL cholesterol levels and coronary heart disease. Carriers of the ApoA-I
Milano
(A-I
M
) mutant have a severe hypoalphalipoproteinemia but are not at increased risk for premature of coronary heart disease. To explain this apparent paradox, we compared the capacity of serum from A-I
M
and control subjects to extract cholesterol from Fu5AH cells. Because the A-I
M
carriers are all heterozygotes for the mutation, we also compared the cholesterol efflux capacity of serum from transgenic mice expressing A-I
M
or wild-type ApoA-I (A-I
WT
), in the absence of murine ApoA-I. In the whole series of human or mouse sera, cholesterol efflux was significantly correlated with several HDL-related parameters; after adjustment for concomitant variables, the only parameter that remained significantly correlated with cholesterol efflux was the serum ApoA-I concentration (
r
2
=0.85 in humans and 0.84 in mice). The same was true when samples from control subjects, A-I
M
carriers, A-I
WT
or A-I
M
mice were analyzed separately. Cholesterol efflux to sera from the A-I
M
carriers was only reduced slightly compared with control sera (25.0±4.2% versus 30.4±3.3%), although there was a large reduction (−45%) in the serum ApoA-I concentration in the former. Cholesterol efflux was also lower to sera from A-I
M
than A-I
WT
mice (15.6±3.8% versus 30.1±7.1%), but less than expected from the 70% reduction in serum ApoA-I concentration. A relative efflux potential of serum was calculated in each group as the slope of the regression line fitting cholesterol efflux to ApoA-I concentrations. Therefore, the relative efflux potential reflects the relative efficiency of ApoA-I in determining cell cholesterol efflux. The relative efflux potential of mouse and human sera was in the following order: A-I
M
mice>A-I
M
carriers>A-I
WT
mice=control subjects, suggesting a gene–dosage effect of the A-I
M
mutation on the efficiency of serum to extract cholesterol from cells. The high efficiency of A-I
M
-containing HDL for cell cholesterol uptake would result in an improved reverse cholesterol transport in the A-I
M
carriers, possibly explaining the low susceptibility to atherosclerosis development.
Enhanced In Vivo Lipid Peroxidation at Elevated Plasma Total Homocysteine Levels
Authors: Voutilainen Sari , Morrow Jason D. , Roberts L. Jackson , Alfthan Georg , Alho Hannu , Nyyssönen Kristiina , Salonen Jukka T.
Published: June 2011

Abstract
—An elevated plasma total homocysteine level (tHcy) is considered an independent risk factor for atherosclerosis. The mechanisms by which hyperhomocysteinemia induces atherosclerosis are only partially understood, but promotion of LDL oxidation and endothelial injury have been suggested. The purpose of this study was to test the hypothesis that a high plasma tHcy is associated in men with increased in vivo lipid peroxidation, as measured by plasma F
2
-isoprostane concentrations. We investigated this association in a subset of the participants in the Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study. Of 256 male participants, a subsample of 100 consecutive men was selected for F
2
-isoprostane assays. The mean tHcy was 11.0 μmol/L, and the mean F
2
-isoprostanes was 29.6 ng/L. The simple correlation coefficient for association between tHcy and F
2
-isoprostane was 0.40 (
P
<0.001). In a linear regression model, the variables with the strongest associations with F
2
-isoprostane were tHcy (standardized coefficient 0.33,
P
<0.001), serum triglycerides (0.21,
P
=0.042), carbohydrate-deficient transferrin (0.15,
P
=0.132), and plasma lipid-standardized α-tocopherol (−0.11,
P
=0.252) (
R
2
=0.24,
P
<0.001 for model). Plasma F
2
-isoprostane levels increased linearly across quintiles of tHcy (
P
<0.001). The unadjusted mean (95% confidence interval) F
2
-isoprostanes was 47.5% greater in the highest tHcy quintile (37.4, 31.1 to 43.6 ng/L) than in the lowest quintile (25.3, 21.3 to 29.3 ng/L). Adjustment for the strongest other determinants of F
2
-isoprostane reduced this difference to 28.2% (
P
=0.010). Our present data suggest that elevated fasting plasma tHcy is associated with enhanced in vivo lipid peroxidation in men.
Effects of a Frequent Apolipoprotein E Isoform, ApoE4
Freiburg
(Leu28→Pro), on Lipoproteins and the Prevalence of Coronary Artery Disease in Whites
Authors: Orth Matthias , Weng Wei , Funke Harald , Steinmetz Armin , Assmann Gerd , Nauck Matthias , Dierkes Jutta , Ambrosch Andreas , Weisgraber Karl H. , Mahley Robert W. , Wieland Heinrich , Luley Claus
Published: June 2011

Abstract
—Different isoforms of apoE modulate the concentrations of plasma lipoproteins and the risk for atherosclerosis. A novel apoE isoform, apoE4
Freiburg
, was detected in plasma by isoelectric focusing because its isoelectric point is slightly more acidic than that of apoE4. ApoE4
Freiburg
results from a base exchange in the
APOE4
gene that causes the replacement of a leucine by a proline at position 28. Analysis of the allelic frequencies in whites in southwestern Germany revealed that this isoform is frequent among control subjects (10:4264 alleles) and is even more frequent in patients with coronary artery disease (21:2874 alleles;
P
=0.004; adjusted odds ratio, 3.09; 95% confidence interval, 1.20 to 7.97). ApoE4
Freiburg
affects serum lipoproteins by lowering cholesterol, apoB, and apoA-I compared with apoE4 (
P
<0.05). Our 4 apoE4
Freiburg
homozygotes suffered from various phenotypes of hyperlipoproteinemia (types IIa, IIb, IV, and V). In vitro binding studies excluded a binding defect of apoE4
Freiburg
, and in vivo studies excluded an abnormal accumulation of chylomicron remnants. ApoE4
Freiburg
and apoE4 accumulated to a similar extent in triglyceride-rich lipoproteins. HDLs, however, contained about 40% less apoE4
Freiburg
than apoE4. In conclusion, our data indicate that apoE4
Freiburg
exerts its possible atherogenic properties by affecting the metabolism of triglyceride-rich lipoproteins and HDL.
Ionizing Radiation Accelerates Aortic Lesion Formation in Fat-Fed Mice via SOD-Inhibitable Processes
Authors: Tribble Diane L. , Barcellos-Hoff Mary Helen , Chu Berbie M. , Gong Elaine L.
Published: June 2011

Abstract—Ionizing radiation promotes formation of reactive oxygen species, including the superoxide anion (O2−). To evaluate whether O2−or O2−-mediated perturbations may contribute to the known atherogenic effects of radiation, we examined aortic lesion formation in irradiated C57BL/6 mice and evaluated the effects of CuZn-superoxide dismutase (CuZn-SOD) overexpression. Ten-week-old mice were exposed to a 2-, 4-, or 8-Gy dose of 250-keV x-rays to the upper thorax and then placed on a high-fat diet for 18 weeks. Based on quantitative lipid staining of serial sections of the proximal aorta, mean lesion area was increased with increasing radiation dose and was 3-fold greater in 8-Gy–irradiated than sham-irradiated mice (7800±2140 versus 2635±709 μm2,P<0.05). These effects were absolutely dependent on a high-fat diet, which had to be introduced within 1 to 2 weeks of the radiation exposure, suggesting the early involvement of atherogenic lipoproteins that were elevated in response to the diet. The importance of radiation-induced oxidative stress was supported by the observation of a 2-fold lower mean lesion area in irradiated CuZn-SOD transgenic mice than in their irradiated, nontransgenic littermates (3026±1590 versus 6102±1834 μm2,P<0.05). Lucigenin-enhanced chemiluminescence, used as an index of aortic O2−concentrations, was significantly elevated in the postradiation period, and this response was reduced in CuZn-SOD transgenics. On the basis of these results, we propose that radiation may be a useful tool for initiating oxidative or redox-regulated events that promote atherogenesis and for testing the antiatherogenic properties of antioxidants.