bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Recovery of Injury-Caused Downregulation of Paxillin Protein by eNOS Gene Expression in Rat Carotid Artery
Authors: Fang Shengyun , Sharma Ram V. , Bhalla Ramesh C.
Published: June 2011

Abstract
—Injury-caused dedifferentiation accompanied by proliferation and migration of smooth muscle cells (SMCs) is an important process in the development of the neointima. Nitric oxide (NO) stimulates differentiation and inhibits proliferation and migration of SMCs. Paxillin has been found to play an important role in cell differentiation, and its phosphorylation is regulated by NO in cultured SMCs. However, the regulation of paxillin by NO in the injured artery has not been investigated. Therefore, the aim of this study was to study the effects of in vivo endothelial NO synthase (eNOS) gene transfection on paxillin expression and intimal hyperplasia. A catheter balloon–denuded rat carotid artery was transfected in vivo with the replication-deficient adenovirus Ad5/RSVeNOS or with Ad5/RSVLacZ as the control. Transfected eNOS gene expression was determined by immunostaining, Western blot analysis, and citrulline assay. The expression of paxillin and its associated proteins was determined in injured arteries by Western blot analysis. The area of the intima and the ratio of intima to media were examined on cross sections by morphometry. The data showed that the expression of paxillin was significantly downregulated after injury. eNOS gene transfer showed no effect on paxillin downregulation 2 days after injury but significantly enhanced the recovery of paxillin protein 5 days and 2 weeks after injury. Vinculin, a paxillin-binding protein, was not altered by vascular injury or by eNOS gene transfer. eNOS gene transfer significantly inhibited intimal hyperplasia for up to 4 weeks. These results suggest that NO inhibition of intimal hyperplasia may be mediated by enhancing the recovery of injury-caused downregulation of paxillin.
Thrombin Activates Factor XI on Activated Platelets in the Absence of Factor XII
Authors: Oliver Julie A. , Monroe Dougald M. , Roberts Harold R. , Hoffman Maureane
Published: June 2011

Abstract
—Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor–initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.
Studies on the Histogenesis of Myxomatous Tissue of Human Coronary Lesions
Authors: Tjurmin Alexey V. , Ananyeva Natalya M. , Smith Elizabeth P. , Gao Yamei , Hong Mun K. , Leon Martin B. , Haudenschild Christian C.
Published: June 2011

Abstract—Myxomatous tissue is a characteristic component of human coronary artery lesions, found more often in restenotic lesions. It represents a bulky accumulation of stellate-shaped cells of unknown histogenesis that are embedded in a loose stroma. We analyzed 64 atherectomy specimens containing substantial amounts of myxomatous tissue by using immunohistochemistry, in situ hybridization, and electron microscopy techniques. Stellate cells represented a heterogeneous population, sharing features of smooth muscle cells (SMCs), macrophages, as well as antigen-presenting dendritic cells. Like quiescent medial SMCs, the stellate cells in all specimens expressed high levels of SM α-actin message and protein and showed heterogeneity with respect to heavy-chain myosin, SM22, desmin, and vimentin. Ultrastructurally, stellate cells resembled SMCs, with some peculiarities that distinguish them from both differentiated and dedifferentiated SMCs. In contrast to quiescent SMCs, the stellate cells expressed high levels of acidic fibroblast growth factor mRNA and protein similar to cells of monocyte/macrophage lineage. However, stellate cells did not express the marker of mature macrophages, HAM56, and were heterogeneous with respect to CD68. Moreover, unlike SMCs, the stellate cells bore some of the major phenotypic markers of dendritic cells: they were S100-positive and showed various reactivity with respect to CD1a and human leukocyte antigen (HLA)-DR. Invasion of myxomatous tissue with CD45RO-positive T lymphocytes was correlated with strong expression of CD1a in these specimens. Stellate cells also expressed a pericyte marker, high-molecular-weight melanoma-associated antigen. We conclude that stellate cells of myxomatous tissue represent a specific phenotype of mesenchymal cells (possibly pericytes), which is activated to express some markers of antigen-presenting cells. These findings suggest involvement of the stellate cells in a local immune response.
Oxidized LDL Can Induce Macrophage Survival, DNA Synthesis, and Enhanced Proliferative Response to CSF-1 and GM-CSF
Authors: Hamilton John A. , Myers Damian , Jessup Wendy , Cochrane Fiona , Byrne Robert , Whitty Genevieve , Moss Suzanne
Published: June 2011

Abstract—Modification of low density lipoprotein (LDL), eg, by oxidation, has been proposed as being important for the formation of foam cells and therefore for the development of atherosclerotic plaques. There are a number of reports showing that macrophage-derived foam cells can proliferate in both human and animal lesions, particularly in the early phase of the disease and possibly involving macrophage-colony stimulating factor (M-CSF, or CSF-1). We studied the in vitro effects of oxidized LDL (ox-LDL) on murine bone marrow–derived macrophages (BMMs), a cell population with a high proliferative capacity in vitro in response to CSF-1 and a dependence for survival on the presence of this growth factor. We report here that treatment of BMMs with low doses of ox-LDL, but not with native LDL, led to cell survival, DNA synthesis, and an enhanced response to the proliferative actions of CSF-1 and granulocyte macrophage-CSF (GM-CSF); the effects were dependent on the degree of LDL oxidation. For CSF-1, a synergistic effect was noticeable at suboptimal doses. The effect of ox-LDL occurred even in the absence of endogenous CSF-1 or GM-CSF. Our findings suggest that ox-LDL, and possibly other modified forms of LDL, could maintain macrophage (and foam cell) survival and therefore lengthen their tenure in a plaque; the modified LDL could also cause local macrophage proliferation or “prime” them so that they could proliferate better in response to CSF-1 (and GM-CSF) concentrations that may be present in the atheroma.
Influence of Elastin Gene Polymorphism on the Elastin Content of the Aorta
Authors: Sauvage Marie , Osborne-Pellegrin Mary , Flohic Françoise Deslandes-Le , Jacob Marie Paule
Published: June 2011

Abstract
—The elastin content in the thoracic aorta of male Brown-Norway (BN) rats is 31.4±1.2% (dry weight), whereas that of male LOU rats is 37.2±1.0%. A similar difference in the elastin content of the thoracic aorta is also observed in female animals. Furthermore, in the thoracic aorta of young, growing rats as well as in cultured aortic smooth muscle cells, the steady-state level of elastin mRNA is significantly lower in the BN than in the LOU strain. These results suggested that 1 or more genes control the elastin mRNA level and the elastin content in the aortas of BN and LOU rats. A possible relationship between a polymorphism in the elastin gene and the elastin content of the aorta was tested. For this purpose, the aortic elastin content was measured in F
1
and F
2
generations bred from LOU and BN rats and was compared with that of the F
0
(parental) generation. A polymorphic marker located in intron 25 of the elastin gene has been used to genotype the F
2
rats. The degree of genetic determination of aortic elastin content was estimated to be 73% in the F
2
cohort, but the elastin locus accounts for only 3.9% of the total variance in aortic elastin content. Other genes are thus responsible for the major part of the observed interstrain difference by regulating the transcription of the gene, the stability of elastin mRNA, and/or posttranslational events.
An In Vitro Coculture Model of Transmigrant Monocytes and Foam Cell Formation
Authors: Takaku Mikio , Wada Youichiro , Jinnouchi Katsunori , Takeya Motohiro , Takahashi Kiyoshi , Usuda Hiroyuki , Naito Makoto , Kurihara Hiroki , Yazaki Yoshio , Kumazawa Yoko , Okimoto Yuko , Umetani Michihisa , Noguchi Noriko , Niki Etsuo , Hamakubo Takao , Kodama Tatsuhiko
Published: June 2011

Abstract—To analyze in vitro the migration of monocytes to the subendothelial space, their differentiation into macrophages, and the subsequent formation of foam cells in vitro, we have developed a 2-coculture system with rabbit aortic endothelial cells (AECs), aortic smooth muscle cells (SMCs), and a mixture of matrix proteins on polyethylene filters in chemotaxis chambers. AECs were seeded on a mixture of type I and IV collagen with or without various types of serum lipoproteins (method 1) or on matrix proteins secreted by SMCs (method 2). In these coculture systems, rabbit AECs can maintain a well-preserved monolayer for up to 2 weeks. When human CD14-positive monocytes were added to the upper medium of the system, with monocyte chemotactic protein-1 treatment ≈60% of the monocytes transmigrated within 24 hours and were retained for up to 7 days, whereas without MCP-1 treatment, <30% of monocytes transmigrated. On day 1, transmigrant monocytes were negative for immunostaining of type I and II macrophage scavenger receptors but by day 3, became positive for scavenger receptors as well as other macrophage markers. When oxidized low density lipoprotein was added to the matrix layer of the method I coculture, on day 4 transmigrant cells exhibited lipid deposit droplets, and by day 7, they had the appearance of typical foam cells. Some of the transmigrant cells recovered in the lower medium on day 7 also appeared to be foam cells, indicating foam cell motility and escape from the coculture layer through the filter. In summary, this coculture system is a useful in vitro tool to dissect the cellular and molecular events that make up the process of foam cell formation.
Vitamin C Protects Human Vascular Smooth Muscle Cells Against Apoptosis Induced by Moderately Oxidized LDL Containing High Levels of Lipid Hydroperoxides
Authors: Siow Richard C. M. , Richards Justin P. , Pedley Kevin C. , Leake David S. , Mann Giovanni E.
Published: June 2011

Abstract
—Vascular cell death is a key feature of atherosclerotic lesions and may contribute to the plaque “necrotic” core, cap rupture, and thrombosis. Oxidatively modified low-density lipoproteins (LDLs) are implicated in the pathogenesis of atherosclerosis, and dietary antioxidants are thought to protect the vasculature against LDL-induced cytotoxicity. Because LDL oxidative modification may vary within atherosclerotic lesions, we examined the effects of defined, oxidatively modified LDL species on human arterial smooth muscle cell apoptosis and the cytoprotective effects of vitamin C. Moderately oxidized LDL (0 to 300 μg protein/mL), which has the highest content of lipid hydroperoxides, induced smooth muscle cell apoptosis within 6 hours, whereas native LDL and mildly and highly oxidized LDL had no effect. Moderately oxidized LDL increased cellular DNA fragmentation, release of fragmented DNA into the culture medium, and annexin V binding and decreased mitochondrial dehydrogenase activity and expression of the antiapoptotic mediator Bcl-x
L
. Treatment of cells with native LDL together with the lipid hydroperoxide 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (HPODE, 200 μmol/L, 6 to 24 hours) also induced apoptotic cell death. Pretreatment of smooth muscle cells with vitamin C (0 to 100 μmol/L, 24 hours) attenuated the cytotoxicity and apoptosis induced by both moderately oxidized LDL and HPODE. Our findings suggest that moderately oxidized LDL, with its high lipid hydroperoxide content, rather than mildly or highly oxidized LDL, causes apoptosis of human smooth muscle cells and that vitamin C supplementation may provide protection against plaque instability in advanced atherosclerosis.
Cholesterol-Induced Changes of Type VIII Collagen Expression and Distribution in Carotid Arteries of Rabbit
Authors: Plenz Gabriele , Dorszewski A. , Völker W. , Ko Y. S. , Severs N. J. , Breithardt G. , Robenek H.
Published: June 2011

Abstract
—Lipoproteins play a major role in cardiovascular disease and atherosclerosis. In the vascular wall, they strongly influence the organization of extracellular matrix. The present study set out to investigate the changes in the extracellular matrix of the vessel wall induced by atherogenic diet, focusing on type VIII collagen, a vascular collagen that has not previously been investigated in detail. The influence of cholesterol diet on the expression, distribution, and deposition of type VIII collagen was examined in carotid arteries of New Zealand White rabbits. Carotid arteries of rabbits receiving diet supplemented with 1% cholesterol for 6 weeks and those on the same regimen followed by normal chow for 1 day, 10 days, 5 weeks, and 12 weeks were studied and compared with controls not exposed to the cholesterol diet. Carotid arteries of normocholesterolemic rabbits contained type VIII collagen–expressing cells in all layers, with focal accumulations of expressing cells in the subendothelial areas, the outer medial zone, and the adventitia. In response to cholesterol diet, type VIII collagen synthesis was reduced in media and adventitia and the distribution patterns changed. Expressing cells were found predominantly in the endothelium, and type VIII collagen accumulated in the intimal space. Immunogold labeling for electron microscopy revealed that type VIII collagen in the intima is associated with microfibrils extending from the internal elastic lamina. Withdrawal of cholesterol resulted in reestablishment of the normal distribution pattern. Northern and Western blot analyses supported the immunoconfocal and in situ hybridization data, demonstrating decreased type VIII collagen expression in response to cholesterol diet and progressive recovery to normal levels with time after withdrawal of cholesterol. Our study demonstrates that type VIII collagen is modulated in the presence of cholesterol. The data indicate that type VIII collagen is specifically remodeled during early experimental atherosclerosis, implying a role for this extracellular matrix component in neointimal growth.
Regulatory Effects of HDL on Smooth Muscle Cell Prostacyclin Release
Authors: Viñals Marisa , Martínez-González José , Badimon Lina
Published: June 2011

Abstract
—One mechanism by which high density lipoproteins (HDLs) exert their protective effect against coronary artery disease could be related to the induction of prostacyclin (PGI
2
) release in the vessel wall. We have recently shown that HDL increases PGI
2
production in rabbit smooth muscle cells (RSMCs) and that this increase is dependent on cyclooxygenase-2 (Cox-2). Here we analyze the mechanism by which rabbit HDL induces PGI
2
release in RSMCs. Our results show that although HDL
2
and HDL
3
share a similar capacity to induce Cox-2 protein levels, HDL
3
stimulates a higher PGI
2
release than does HDL
2
, probably because of their relative arachidonate contents. Acetylsalicylic acid pretreatment (300 μmol/L, 30 minutes) significantly reduced the HDL-induced PGI
2
release, suggesting that both preexisting and induced Cox-2 activities were involved in the HDL effect. Ca
2+
-dependent cytosolic phospholipase A
2
(cPLA
2
) and Cox-1 protein levels were not altered by HDL. Dexamethasone (2 μmol/L), which also inhibited the HDL-induced PGI
2
release, reduced significantly both Cox-2 mRNA and protein levels without affecting cPLA
2
and Cox-1 protein levels. In addition, methylarachidonyl fluorophosphonate, a potent inhibitor of cPLA
2
, did not produce any effect on HDL-induced PGI
2
release. In the presence of cycloheximide, Cox-2 mRNA levels were induced by HDL and inhibited by dexamethasone, suggesting that HDL and dexamethasone work in the absence of de novo protein synthesis. These results indicate an early effect of HDL on PGI
2
biosynthesis, specifically increasing Cox-2. PD98059, an inhibitor of mitogen-activated protein kinase kinase, completely inhibited HDL-induced PGI
2
release, whereas GF109203X, a protein kinase C inhibitor, had no effect. Thus, HDL induces PGI
2
synthesis by a mechanism dependent on the mitogen-activated protein kinase pathway but independent of protein kinase C.
LDL Particle Size Distribution Is Associated With Carotid Intima-Media Thickness in Healthy 50-Year-Old Men
Authors: Skoglund-Andersson Camilla , Tang Rong , Bond M. Gene , de Faire Ulf , Hamsten Anders , Karpe Fredrik
Published: June 2011

Abstract
—Results of cross-sectional and prospective studies have suggested that small, dense low-density lipoprotein (LDL) particles predispose to coronary heart disease. We investigated the relationships between plasma concentrations of LDL subfractions and intima-media thickness (IMT) of the common carotid artery (CCA), quantified by B-mode ultrasound, in 94 healthy, 50-year-old men, all of whom were homozygous for the apolipoprotein E3 allele. A novel 3% to 7.5% polyacrylamide gradient gel was developed to provide separation of LDL subfractions with high resolution, as was a procedure to quantify plasma concentrations of these LDL subspecies. The LDL particle size distribution pattern obtained by the gradient gel electrophoresis procedure was in good agreement with the one obtained by a well-established, single-spin density gradient ultracentrifugation technique. LDL-II (particle size, 23.5 to 25.0 nm) was the most abundant subfraction, and its plasma concentration correlated closely with the total LDL cholesterol concentration (
r
=0.61,
P
<0.001) but not with CCA IMT (
r
=−0.13, NS). In contrast, the plasma concentration of the predominant small, dense LDL particle subfraction (LDL-III; particle size, 22.5 to 23.5 nm) correlated strongly with CCA IMT (
r
=0.42,
P
<0.001). In multivariate analysis, the plasma concentration of the LDL-III subfraction contributed significantly to the variation in CCA IMT (
R
2
=0.19). When plasma triglycerides and LDL cholesterol were forced into the multivariate model, 10% of the variation in CCA IMT was still accounted for by the LDL-III subfraction. In summary, use of a novel and sensitive gradient gel electrophoresis method for evaluation of LDL heterogeneity provided the basis for demonstrating an independent relation between the plasma concentration of small LDL and IMT of the CCA in healthy, middle-aged men.
Independent Effects of Apo E Phenotype and Plasma Triglyceride on Lipoprotein Particle Sizes in the Fasting and Postprandial States
Authors: Dart Anthony M. , Cooper Bridget
Published: June 2011

Abstract
—LDL particle sizes and Apo E phenotypes were determined in 212 subjects of whom 51 had angina. LDL diameter was significantly less in subjects with an ε2 allele (24.76±0.08 vs 24.94±0.02 nm,
P
=0.02), and this was evident for both E2/E3 (24.77±0.09 nm) and E2/E4 (24.69±0.08 nm) phenotypes. Although there was a negative relation between LDL diameter and plasma triglyceride, the effect of apo E2 was still evident with adjustment for triglyceride. In multiple regression analysis, the significant determinants of LDL diameter were gender (with females having larger particles than males), body mass index, and the presence (or absence) of E2. HDL particle sizes and compositions were determined on fasting samples and, additionally, 5 and 8 hours after a fat-rich meal for 48 coronary heart disease cases and 49 control subjects. Fasting HDL particle sizes were not related to the presence of E2 but were significantly smaller for subjects possessing an ε4 allele (8.09±0.08 vs 8.39±0.05 nm,
P
=0.003) and were negatively related to plasma triglyceride. However, the effect of E4 persisted after adjustment for triglyceride. In a multiple regression analysis, the only significant determinant of fasting HDL diameter was the presence (or absence) of E4 with fasting plasma triglyceride just failing to reach significance (
P
=0.06). There was a postprandial increase in HDL diameter that was less marked in subjects with coronary heart disease. The postprandial increase in HDL diameter was of sufficient magnitude to result in size reclassification of HDL particles. The influence of E4 was also evident at both postprandial time points. Compositional analysis demonstrated that the increase in HDL diameters postprandially could be attributed to triglyceride enrichment, with an accompanying fall in cholesterol ester content. Phospholipid changes postprandially were biphasic with an initial fall followed by a rise in concentration. The increase in triglyceride content was significantly less in those subjects with angina despite an equivalent rise in plasma triglyceride. The present study demonstrates significant, but different, effects of variation in apo E phenotype on the particle sizes of both HDL and LDL. Such effects were still evident with adjustment for differences in plasma triglyceride and suggests that variation in apo E phenotype exerts effects on lipoprotein particle sizes by mechanisms additional to those dependent on change in plasma triglyceride.
Westernization of Chinese Adults and Increased Subclinical Atherosclerosis
Authors: Woo Kam S. , Chook Ping , Raitakari Olli T. , McQuillan Brendan , Feng Jiang Zheng , Celermajer David S.
Published: June 2011

Abstract
—Cardiovascular event rates are much lower in China compared with developed countries. “Westernization” of diet and lifestyle in the Chinese, however, may lead to an increased prevalence of atherosclerosis-related diseases. Because carotid intima-media thickness (IMT) is a marker of subclinical atherosclerosis, we examined IMT and vascular risk profile in community-based groups of rural Chinese, Westernized urban Chinese, and urban whites. Mean IMT of the common carotid artery was measured in 348 healthy adults, aged 42±13 years (range 21 to 71 years); 116 subjects from rural China, 116 urban Chinese subjects living in Hong Kong or in Australia, and 116 urban Caucasians living in Australia. These 3 groups were matched for age, sex, and cigarette smoke exposure. Urban Chinese subjects had slightly better risk factor profile (higher HDL-cholesterol and lower blood pressure) compared with rural Chinese subjects. Despite this, however, the mean IMT was lowest in rural Chinese (0.50±0.10 mm), intermediate in urban Chinese (0.56±0.12 mm), and highest in urban whites (0.64±0.13 mm) (
P
<0.001 for comparisons between all groups). These differences in IMT were not altered after adjustment for the major traditional cardiovascular risk factors (serum lipids, smoking, and blood pressure or for body mass index). The influence of vascular risk factors on atherosclerosis between urban versus rural Chinese subjects was studied by multivariate regression models and by comparing the steepness of regression slopes between risk factors and IMT in the subject groups. The effects of smoking, HDL-cholesterol, and triglycerides on IMT were significantly greater in the urban compared with the rural Chinese (
P
<0.01). These data suggest that Westernization of Chinese subjects is associated with greater susceptibility to the pro-atherogenic effects of traditional vascular risk factors, such as lipids and smoking, and with evidence of increased IMT as a marker of subclinical atherosclerosis.
Anderson’s Disease
Authors: Dannoura A. Hayssam , Berriot-Varoqueaux Nathalie , Amati Patricia , Abadie Veronique , Verthier Nicole , Schmitz Jacques , Wetterau John R. , Samson-Bouma Marie-Elisabeth , Aggerbeck Lawrence P.
Published: June 2011

Abstract
—Anderson’s disease is a rare, hereditary hypocholesterolemic syndrome characterized by chronic diarrhea, steatorrhea, and failure to thrive associated with the absence of apo B48–containing lipoproteins. To further define the molecular basis of the disease, we studied 8 affected subjects in 7 unrelated families of North African origin after treatment with a low-fat diet. Lipid loading of intestinal biopsies persisted, but the pattern and extent of loading was variable among the patients. Electron microscopy showed lipoprotein-like particles in membrane-bound compartments, the densities (0.65 to 7.5 particles/μ
2
) and the mean diameters (169 to 580 nm) of which were, in general, significantly larger than in a normal fed subject (0.66 particles/μ
2
, 209 nm mean diameter). There were also large lipid particles having diameters up to 7043 nm (average diameters from 368 to 2127 nm) that were not surrounded by a membrane. Rarely, lipoprotein-like particles 50 to 150 nm in diameter were observed in the intercellular spaces. Intestinal organ culture showed that apo B and apo AIV were synthesized with apparently normal molecular weights and that small amounts were secreted in lipid-bound forms (density <1.006 g/mL). Normal microsomal triglyceride transfer protein (MTP) and activity were also detected in intestinal biopsies. Segregation analyses of 4 families excluded, as a cause of the disease, significant regions of the genome surrounding the genes for apo AI, AIV, B, CI, CII, CIII, and E, as were the genes encoding 3 proteins involved in intracellular lipid transport, MTP, and fatty acid binding proteins 1 and 2. The results suggest that a factor other than apoproteins and MTP are important for human intestinal chylomicron assembly and secretion.
Prothrombin G20210A Gene Mutation and Further Prothrombotic Risk Factors in Childhood Thrombophilia
Authors: Junker Ralf , Koch Hans-Georg , Auberger Karin , Münchow Nicole , Ehrenforth Silke , Nowak-Göttl Ulrike
Published: June 2011

Abstract
—Risk factors for venous thrombosis in adults are the prothrombin G20210A and the factor V (FV) G1691A mutations and hereditary deficiencies of protein C, protein S and antithrombin. However, data are limited on the relevance of these risk factors for thrombosis in children and adolescents. We therefore investigated 261 patients aged 0 to 18 (median 5.7 years, 48% male) with venous thrombosis and controls (n=370) for the presence of prothrombotic risk factors including the prothrombin G20210A mutation. The following frequencies of hereditary risk factors (patients versus controls), odds ratios (OR) and 95% confidence intervals (CI), or results of Fisher’s exact test, respectively, were found: prothrombin G20210A, 4.2% versus 1.1%, OR/CI 4.1/1.3 to 12.8; FV G1691A, 31.8% versus 4.1%, OR/CI 11.0/6.2 to 19.7; protein C deficiency, 9.2% versus 0.8%, OR/CI 12.4/3.7 to 41.6, protein S deficiency, 5.7% versus 0.8%, OR/CI 7.5/2.1 to 26.0; antithrombin deficiency in 3.4% in the patients, but not in the controls,
P
=0.0003. The prothrombin mutation was combined with the heterozygous FV G1691A mutation (2.3%) or protein C deficiency (0.3%) in the patients, but not in the controls (prothrombin and FV mutation,
P
=0.0048; prothrombin and protein C deficiency, not significant). The carrier frequencies and ORs of all hereditary risk factors showed a non-significant trend toward higher prevalences in patients suffering spontaneous thrombosis, compared with those with an additional underlying disease. In conclusion, the prothrombin G20210A and the FV G1691A mutation, deficiencies of protein C, protein S, and antithrombin are important risk factors for venous thrombosis during childhood and adolescence.
Characterization of an E-box–Dependent cis Element in the Smooth Muscle α-Actin Promoter
Authors: Jung Frank , Johnson A. Daniel , Kumar Meenakshi S. , Wei Beiyang , Hautmann Martina , Owens Gary K. , McNamara Coleen
Published: June 2011

Abstract
—Identification of the regulators of smooth muscle specific gene expression is critical for understanding smooth muscle cell (SMC) differentiation and the alterations in SMC phenotype seen in vascular diseases. Previous studies have identified that a 2-bp mutation in a conserved cis-acting element (TGTTTATC) in the promoter of the chicken smooth muscle (SM) α-actin gene abolished nuclear factor binding and decreased transcriptional activity of a 271-bp SM α-actin promoter fragment when transfected into rat aortic SMC. However, the promoter region containing this conserved sequence has negative cis regulatory activity when studied in homologous systems. The goal of the present studies was to further characterize the transcriptional activity of the rat SM α-actin promoter region between –224 and –236 that is conserved across mammals. DNAse I analysis and electrophoretic mobility shift assays demonstrated that SMC nuclear proteins bound an extended sequence (TGTTTATCCCCATAA). Transient transfection experiments of wild-type and mutant rat SM α-actin promoter-luciferase constructs into rat aortic SMC revealed that promoter activity was enhanced by mutations of specific nucleotides in the TGTTTATCCCCA region. Interestingly, the TGTTTATCCCCA element in the rat SM α-actin promoter is centered between 2 canonical E-boxes. Mutations of the flanking E-boxes abolished the enhancement in promoter activity seen with mutation of the TGTTTATCCCCA element alone. Thus studies provide evidence for a regulatory cassette in the rat SM α-actin promoter that regulates gene expression via combinatorial interactions between 2 E-boxes and a newly described TGTTTATCCCCA element.
Chlamydia pneumoniae
in Abdominal Aortic Aneurysms
Authors: Meijer Adam , van der Vliet J. Adam , Roholl Paul J. M. , Gielis-Proper Siska K. , de Vries Ankje , Ossewaarde Jacobus M.
Published: June 2011

Abstract
—In this article, we describe the results of a comparative study for the detection of
Chlamydia pneumoniae
in abdominal aortic aneurysm specimens of 19 patients through the use of immunocytochemistry (ICC), in situ hybridization (ISH), and polymerase chain reaction (PCR), along with the detection of cytomegalovirus (CMV) and herpes simplex virus (HSV) by ICC and PCR.
C pneumoniae
–specific membrane protein was detected in specimens of all 19 (100%; 95% confidence interval [CI] 82% to 100%) and of 15 (79%; 95% CI 54% to 94%) patients with monoclonal antibodies RR-402 and TT-401, respectively. Chlamydial lipopolysaccharide was detected in specimens of 15 (79%; 95% CI 54% to 94%) patients when the results of 4 different monoclonal antibodies were combined. Surprisingly, chlamydial heat shock protein 60 was not detected in any of the specimens by ICC. Furthermore,
C pneumoniae
DNA was not detected by ISH when a
C pneumoniae
major outer membrane protein gene fragment was used as probe, nor was it reproducibly detected by PCR on extracted DNA. These results may be explained either by different kinetics of degradation of the different components of
C pneumoniae
after infection of the vessel wall or by the involvement of other
Chlamydia
-like microorganisms. Coexistence of
C pneumoniae
antigens and HSV antigens but not CMV antigens was observed in specimens from 10 of 18 (56%; 95% CI 31% to 78%) patients by ICC. CMV and HSV DNAs were not detected by PCR. In conclusion, we have demonstrated the presence of antigens of
C pneumoniae
in the absence of specific DNA in abdominal aortic aneurysms, suggesting persistence of the antigens rather than a persistent infection.
Expression of LR11, a Mosaic LDL Receptor Family Member, Is Markedly Increased in Atherosclerotic Lesions
Authors: Kanaki Tatsuro , Bujo Hideaki , Hirayama Satoshi , Ishii Itsuko , Morisaki Nobuhiro , Schneider Wolfgang Johann , Saito Yasushi
Published: June 2011

Abstract
—Receptors belonging to the LDL receptor (LDLR) family are thought to play key roles in lipoprotein metabolism in a variety of tissues, including the arterial wall. Here, we report that the expression of a 250-kDa mosaic LDLR family member, which we called LR11 for the presence of 11 ligand-binding repeats, is markedly induced during the process of atherogenesis in 2 animal models. Analysis by reverse transcription–polymerase chain reaction and RNase protection assays revealed that LR11 transcript levels rise in rabbit aortas displaying atheromatous lesions after the rabbits have been fed a high-cholesterol diet. Immunohistochemistry demonstrated that the highest induction of LR11 occurs in intimal smooth muscle cells (SMCs), followed by medial SMCs close to the intimal border of the atheromatous lesions. Experimental intimal hyperplasia by endothelial denudation showed that LR11 mRNA levels were also increased in the arteries after balloon injury, with the transcripts localized primarily in the hyperplastic intimal layer. In agreement with the correlation of LR11 induction during increased cell proliferation, cultured SMCs showed an increase in LR11 expression in the proliferative phase. Furthermore, Northern and Western blot analyses showed that medium conditioned by the monocyte-macrophage cell line THP-1 enhanced LR11 expression in cultured SMCs. These findings suggest that upregulation of LR11 might be contributing to the pathological roles of intimal and medial SMCs during arteriosclerotic lesion development and provide the first insight into the as yet unknown functional significance of this intriguing LDLR family member.
A Hepatic Lipase Gene Promoter Polymorphism Attenuates the Increase in Hepatic Lipase Activity With Increasing Intra-abdominal Fat in Women
Authors: Carr Molly C. , Hokanson John E. , Deeb Samir S. , Purnell Jonathan Q. , Mitchell Ellen S. , Brunzell John D.
Published: June 2011

Abstract
—High hepatic lipase (HL) activity is associated with an atherogenic lipoprotein profile of small, dense LDL particles and lower HDL
2
-C. Intra-abdominal fat (IAF) is positively associated with HL activity. A hepatic lipase gene (
LIPC
) promoter variant (G→A
−250
) is associated with lower HL activity, higher HDL
2
-C, and less dense LDL particles. To determine whether the
LIPC
promoter polymorphism acts independently of IAF to regulate HL, 57 healthy, premenopausal women were studied. The
LIPC
promoter A allele was associated with significantly lower HL activity (GA/AA=104±34 versus GG=145±57 nmoles · mL
−1
· min
−1
,
P
=0.009). IAF was positively correlated with HL activity (
r
=0.431,
P
<0.001). Multivariate analysis revealed a strong relationship between both the
LIPC
promoter genotype (
P
=0.001) and IAF (
P
<0.001) with HL activity. The relationship between IAF and HL activity for carriers and noncarriers of the A allele was curvilinear with the carriers having a lower apparent maximum level of plasma HL activity compared with noncarriers (138 versus 218 nmoles · mL
−1
· min
−1
,
P
<0.001). In addition, the
LIPC
A allele was associated with a significantly higher HDL
2
-C (GA/AA=16±7 versus GG=11±5 mg/dL,
P
=0.003). We conclude that the LIPC promoter A allele attenuates the increase in HL activity due to IAF in premenopausal women.
Decreased Production Rates of VLDL Triglycerides and ApoB-100 in Subjects Heterozygous for Familial Hypobetalipoproteinemia
Authors: Elias Nizar , Patterson Bruce W. , Schonfeld Gustav
Published: June 2011

Abstract
—Familial hypobetalipoproteinemia (FHBL) is an autosomal codominant disorder characterized by low levels of apolipoprotein (apo) B and low-density lipoprotein (LDL) cholesterol. Decreased production rates of apoB have been demonstrated in vivo in FHBL heterozygotes. In the present study, we wished to investigate whether the transport of triglycerides was similarly affected in these subjects. Therefore, we studied the in vivo kinetics of very-low-density lipoprotein (VLDL) triglycerides and VLDL apoB-100 simultaneously in 7 FHBL heterozygotes from 2 well-characterized kindreds and 7 healthy normolipidemic subjects. In both kindreds, hypobetalipoproteinemia is caused by mutations in the 5′ portion of the apoB gene specifying short truncations of apoB undetectable in plasma. A bolus injection of deuterated palmitate and a primed constant infusion of deuterated leucine were given simultaneously, and their incorporation into VLDL triglycerides and VLDL apoB, respectively, were determined by gas chromatography–mass spectrometry. Kinetic parameters were calculated by using compartmental modeling. VLDL apoB fractional catabolic rates (FCRs) in FHBL heterozygotes and controls were similar (11.6±3.9 and 10.9±2.4 pools per day, respectively,
P
=0.72). On the other hand, FHBL heterozygotes had a 75% decrease in VLDL apoB production rates compared with normal subjects (5.8±1.8 versus 23.4±7.1 mg/kg per day,
P
<0.001). The decreased production rates of VLDL apoB accounts for the very low concentrations of plasma apoB found in heterozygotes from these kindreds (24% of normal). Mean VLDL triglyceride FCRs in FHBL subjects and controls were not significantly different (1.06±0.74 versus 0.89±0.50 pools per hour, respectively,
P
=0.61). There was a good correlation between VLDL apoB FCR and VLDL triglyceride FCR in the 2 groups (
r
=0.84,
P
<0.001). VLDL triglyceride production rates were decreased by 60% in FHBL heterozygotes compared with controls (9.3±6.0 versus 23.0±9.6 μmol/kg per hour,
P
=0.008). Thus, the hepatic secretion of VLDL triglycerides is reduced in FHBL heterozygotes but to a lesser extent than the decrease in apoB-100 secretion. This is probably achieved by the secretion of VLDL particles enriched with triglycerides.
Increased Prevalence of Smaller and Denser LDL Particles in Asian Indians
Authors: Kulkarni Krishnaji R. , Markovitz Jerome H. , Nanda Navin C. , Segrest Jere P.
Published: June 2011

Abstract
—There is increasing evidence to believe that Asian Indians are at an increased risk of coronary heart disease (CHD), which cannot be attributed to the common risk factors. Individuals with small, dense LDL phenotype are also known to be at increased risk of CHD. Our objective was to examine whether the prevalence of smaller and denser LDL particles is increased in Asian Indians. Thirty-nine Asian Indians (22 men and 17 women), aged 25 to 45 years, were matched with 39 whites for age and gender. Cholesterol profiles of lipoprotein classes and LDL subclasses were measured using the Vertical Auto Profile–II (VAP-II) and LDL-VAP-II methods, respectively. Six LDL subclasses (LDL1 to LDL6) have been identified using the LDL-VAP-II, with LDL1 and LDL6, respectively, being the most and least buoyant subclasses. The prevalence of small, dense LDL type (subjects with major LDL subclass 5 or 6) was significantly higher in Asian Indians compared with white subjects (44% versus 21%;
P
<0.05). The relative position of the major LDL density peak (LDL-Rf) on 0 to 1 scale in LDL-VAP-II density gradient was also significantly decreased in Asian Indians (0.462±0.076 versus 0.505±0.086;
P
<0.02), suggesting an increased LDL density. Furthermore, this increased prevalence of small, dense LDL type appears to be due to the increased triglycerides (TG) (r for LDL-Rf versus TG=0.681,
P
<0.001), with fasting insulin being one of the important determinants of TG (r for TG versus fasting insulin=0.572,
P
<0.001). In addition, fasting insulin was significantly increased in Asian Indians with small, dense LDL type compared with other Asian Indians, suggesting a significant role of insulin resistance in increasing the prevalence of small, dense LDL type. We conclude that the increased prevalence of small, dense LDL observed in Asian Indians might contribute to their increased CHD risk.
Correlation Between Flow-Mediated Vasodilatation of the Brachial Artery and Intima-Media Thickness in the Carotid Artery in Men
Authors: Hashimoto Masayoshi , Eto Masato , Akishita Masahiro , Kozaki Koichi , Ako Junya , Iijima Katsuya , Kim Seungbum , Toba Kenji , Yoshizumi Masao , Ouchi Yasuyoshi
Published: June 2011

Abstract
—Endothelial dysfunction has been reported to be the initial step in atherosclerosis. A noninvasive technique that uses ultrasound to measure the intima-media thickness of the carotid artery has been applied to evaluate localized atherosclerosis. This study was undertaken to elucidate whether endothelial dysfunction in the brachial artery is related to the intima-media thickness of the carotid artery. Thirty-four men with atherosclerosis (mean±SE age 61±2 years) and 33 age-matched men without clinical atherosclerosis were examined. The intima-media thickness and plaque formation of the common carotid artery were assessed by B-mode ultrasonography. We also noninvasively measured brachial artery diameter by the same ultrasound machine when the subjects were at rest, during reactive hyperemia, which causes endothelium-dependent vasodilatation, and after sublingual administration of nitroglycerin, which causes endothelium-independent vasodilatation. The atherosclerosis group had a significantly greater intima-media thickness of the common carotid artery than did the control group (1.02±0.04 versus 0.91±0.03 mm,
P
<0.05). The flow-mediated diameter (FMD) increase (percent FMD=ΔD/D×100) in the atherosclerosis group was significantly smaller than that in the control group (2.8±0.4% versus 5.1±0.6%,
P
<0.01). A significant negative correlation between the intima-media thickness of the carotid artery and percent FMD was found in all of the subjects. On multiple regression analysis, percent FMD showed a significant negative correlation with the intima-media thickness of the common carotid artery. These findings support the concept that endothelial dysfunction is significantly related to atherogenesis.
Tissue Plasminogen Activator (tPA) Deficiency Exacerbates Cerebrovascular Fibrin Deposition and Brain Injury in a Murine Stroke Model
Authors: Tabrizi Peyman , Wang Liang , Seeds Nicholas , McComb J. Gordon , Yamada Shinya , Griffin John H. , Carmeliet Peter , Weiss Martin H. , Zlokovic Berislav V.
Published: June 2011

Abstract
—Although the serine protease, tissue plasminogen activator (tPA), is approved by the US Food and Drug Administration for therapy to combat focal cerebral infarction, the basic concept of thrombolytic tPA therapy for stroke was challenged by recent studies that used genetically manipulated tPA-deficient (tPA−/−) mice, which suggested that tPA mediates ischemic neuronal damage. However, those studies were potentially flawed because the genotypes of tPA−/− and wild-type control mice were not entirely clear, and ischemic neuronal injury was evaluated in isolation of tPA effects on brain thrombosis. Using mice with appropriate genetic backgrounds and a middle cerebral artery occlusion stroke model with nonsiliconized thread, which does lead to microvascular thrombus formation, in the present study we determined the risk for cerebrovascular thrombosis and neuronal injury in tPA−/− and genetically matched tPA+/+ mice subjected to transient focal ischemia. Cerebrovascular fibrin deposition and the infarction volume were increased by 8.2- and 6.7-fold in tPA−/− versus tPA+/+ mice, respectively, and these variables were correlated with reduced cerebral blood flow up to 58% (
P
<0.05) and impaired motor neurological score by 70% (
P
<0.05). Our findings indicate that tPA deficiency exacerbates ischemia-induced cerebrovascular thrombosis and that endogenous tPA protects the brain from an ischemic insult, presumably through its thrombolytic action. In addition, our study emphasizes the importance of appropriate genetic controls in murine stroke research.
Inhibition of Tissue Factor Pathway During Intermittent Pneumatic Compression
Authors: Chouhan Vibhuti D. , Comerota Anthony J. , Sun Ling , Harada Russell , Gaughan John P. , Rao A. Koneti
Published: June 2011

Abstract
—Intermittent pneumatic compression (IPC) devices are an effective prophylaxis against lower extremity deep vein thrombosis. Their antithrombotic effect has been attributed to a reduction in venous stasis and enhanced fibrinolysis. The initiating mechanism for blood coagulation is the tissue factor (TF) dependent pathway, which is inhibited by tissue factor pathway inhibitor (TFPI). We have investigated the effect of IPC on the TF pathway in 6 normal subjects and 6 patients with postthrombotic venous disease undergoing IPC for 120 minutes; all subjects were studied with each of 5 IPC devices. In normal subjects and patients, plasma factor VIIa (FVIIa) activity (the activated form of factor VII [FVII]) declined from mean values ranging 51 to 65 and 50 to 53 mU/mL before IPC with different devices to 10 to 13 and 20 to 22 mU/mL at 180 minutes, respectively (
P
<0.001 for all groups). FVII antigen levels were unchanged. Plasma TFPI (
P
<0.001) rose from mean baseline values ranging 69 to 79 and 57 to 61 ng/mL to 76 to 123 and 71 to 79 ng/mL at 180 minutes in normal subjects and patients, respectively (
P
<0.001 for all groups). Plasma prothrombin fragment F1.2 levels showed minimal changes. There was an inverse relationship between TFPI and FVIIa in normal subjects (
r
=−0.31,
P
=0.001) and patients (
r
=−0.37,
P
<0.001). IPC results in an increase in plasma TFPI and decline in FVIIa. Inhibition of TF pathway, the initiating mechanism of blood coagulation, is a possible mechanism for the antithrombotic effect of IPC.
Physiological Increments in Plasma Homocysteine Induce Vascular Endothelial Dysfunction in Normal Human Subjects
Authors: Chambers John C. , Obeid Omar A. , Kooner Jaspal S.
Published: June 2011

Abstract
—We hypothesized that physiological increments in plasma homocysteine after low-dose oral methionine or dietary animal protein induce vascular endothelial dysfunction and that there is a graded, inverse relationship between homocysteine concentration and endothelial function. We studied 18 healthy volunteers aged 18 to 59 years. Brachial artery flow-mediated and glyceryltrinitrate-induced dilatation were measured after 1) oral L-methionine (10, 25, and 100 mg/kg), 2) dietary animal protein (lean chicken 551±30 g, comprising 3.2±0.2 g methionine), and 3) methionine-free amino acid mix (100 mg/kg). Methionine (10, 25, and 100 mg/kg) induced a dose-related increase in homocysteine (9.4±1.3 to 12.2±2.1, 17.6±2.6, and 26.1±4.2 μmol/L, respectively;
P
<0.001) and a reduction in flow-mediated dilatation (4.1±0.8 to 2.1±0.8, 0.3±0.8, and −0.7±0.8%, respectively;
P
<0.001) at 4 hours. Compared with usual meal, animal protein increased plasma homocysteine (9.6±0.8 to 11.2±0.9 μmol/L,
P
=0.005) and reduced flow-mediated dilatation (4.5±0.7% to 0.9±0.6%,
P
=0.003). Methionine-free amino acid mix did not induce any changes. Glyceryltrinitrate-induced dilatation was unchanged throughout. In this study, small physiological increments in plasma homocysteine after low-dose methionine and dietary animal protein induced vascular endothelial dysfunction. We propose that protein intake–induced increments in plasma homocysteine may have deleterious effects on vascular function and contribute to the development and progression of atherosclerosis.