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Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647-labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation. © 2012 Society for Laboratory Automation and Screening.

During pregnancy the walls of decidual spiral arteries (SAs) undergo clinically important structural modifications crucial for embryo survival/growth and maternal health. However, the mechanisms of SA remodeling (SAR) are poorly understood. Although an important prerequisite to this understanding is knowledge about the phenotype of SA muscular wall prior to and during the beginning of mouse SAR, this remains largely unexplored and was the main aim of this work. Using histological and immunohistochemical techniques, this study shows for the first time that during early mouse gestation, from embryonic day 7.5 (E7.5) to E10.5, the decidual SA muscular coat is not a homogeneous structure, but consists of two concentric layers. The first is a largely one cell-thick sub-endothelial layer of contractile mural cells (positive for α-smooth muscle actin, calponin and SM22α) with pericyte characteristics (NG2 positive). The second layer is thicker, and evidence is presented that it may be of the synthetic/proliferative smooth muscle phenotype, based on absence (α-smooth muscle actin and calponin) or weak (SM22α) expression of contractile mural cell markers, and presence of synthetic smooth muscle characteristics (expression of non-muscle Myosin heavy chain-IIA and of the cell proliferation marker PCNA). Importantly, immunohistochemistry and morphometrics showed that the contractile mural cell layer although prominent at E7.5-E8.5, becomes drastically reduced by E10.5 and is undetectable by E12.5. In conclusion, this study reveals novel aspects of the decidual SA muscular coat phenotype prior to and during early SAR that may have important implications for understanding the mechanisms of SAR. © 2011 Elsevier Inc. 416 211 216 Cited By :8

Increase in carotid artery intima-media thickness (IMT) is an early sign of atherosclerosis. Slow coronary flow (SCF) is characterized by delay of opacification of coronary arteries in coronary angiography in the absence of any evident obstructive lesion, but its etiopathogenesis remains unclear. Genes that regulate the renin angiotensin system also play a role in developing cardiovascular system disorders. The presence of deletion (D) allele in angiotensin converting enzyme (ACE) gene polymorphism is associated with coronary artery disease. The aim of this study was to investigate the carotid artery IMT measurement, as an early sign of atherosclerosis, in patients with SCF and without SCF and also to assess the effect of the renin-angiotensin gene system on carotid IMT. Forty-four patients with angiographically proven SCF and 44 cases with normal coronary flow (NCF) pattern with similar risk profile were enrolled in the study. Coronary flow patterns of the cases were determined by thrombolysis in myocardial infarction (TIMI) frame count method. Intima-media thickness was measured by recording ultrasonographic images of both the left and right common carotid artery with a 12-MHz linear array transducer. ACE I/D polymorphism and Angiotensin II tip 1 receptor (AT1R) A/C gene polymorphism were determined by polymerase chain reaction (PCR) amplification. Demographic characteristics and coronary artery disease risk factors of SCF and NCF groups were similar. Mean TIMI frame count and carotid IMT (mm) were significantly higher in the SCF group than controls (45.9 ± 12 vs 23.3 ± 3.7, P = 0.0001; 0.75 ± 0.08 vs 0.69 ± 0.06, P = 0.0001, respectively). Mean TIMI frame count was positively correlated with IMT of carotid artery in correlation analysis (r = 0.45, P = 0.0001). When analyzed in regard to ACE genotype in all subjects, IMT values were statistically different (0.78 ± 0.06 for DD genotype, 0.72 ± 0.05 for ID genotype, and 0.64 ± 0.06 for II genotype, P = 0.0001). This difference remained significant in subgroup analyses for each genotype. No association could be observed between the AT1R A/C1166 polymorphism and IMT of carotid artery measurement (P > 0.05). Lack of association was still observed with analysis carried out when genotype effect was assumed to be inherited as additive (CC versus AA versus AC) or dominant (AA versus AC+CC). Increased IMT in patients with SCF shows that subclinical atherosclerosis may play role in this phenomenon. This increase was most marked in the presence of D allele of ACE genotype, which is associated with vascular hypertrophy. © Springer-Verlag Tokyo 2007.

The objective of this study was to investigate differences in intima-media thickness (IMT) and diameter (D) measurements of the common carotid artery (CCA) in ultrasound imaging in normal subjects and renal failure disease (RFD) patients. Manual measurements by two experts and automated segmentation measurements (based on snakes and active contour models (ACM)) were carried out on 73 normal subjects, and 80 RFD patients. Statistical analysis was carried out using the Wilcoxon rank-sum test at p

The cerebral vasculature ensures proper cerebral function by transporting oxygen, nutrients, and other substances to the brain. Distribution of oxygenated blood throughout the neuroaxis takes place at the level of the circle of Willis (CW). While morphologic and functional alterations in CW arteries and its main branches have been reported in cerebrovascular and neurodegenerative diseases, accompanying changes in protein expression profiles remain largely uncharacterized. In this study, we performed proteomics to compile a novel list of proteins present in mouse CW arteries and its ramifications. Circle of Willis arteries were surgically removed from 6-month-old wild-type mice, proteins extracted and analyzed by two proteomics approaches, gel-free nanoLC-mass spectrometry (MS)/MS and gel-based GelLC-MS/MS, using nanoAcquity UPLC coupled with ESI-LTQ Orbitrap XL. The two approaches helped maximize arterial proteome coverage. Six biologic and two technical replicates were performed. In all, 2,188 proteins with at least 2 unique high-scoring peptides were identified (6,630 proteins total). Proteins were classified according to vasoactivity, blood-brain barrier specificity, tight junction and adhesion molecules, membrane transporters/channels, and extracellular matrix/basal lamina proteins. Furthermore, we compared the identified CW arterial proteome with the published brain microvascular proteome. Our database provides a vital resource for the study of CW cerebral arterial protein expression profiles in health and disease. © 2014 ISCBFM All rights reserved.

Objective : To determine the relationship of serum total homocysteine (tHcy), serum folate and 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C→T genotype with ultrasonic arterial wall measurements associated with subclinical atherosclerosis. Study design : Cross-sectional analysis of 767 participants in an ongoing prospective study. Intima-media thickness (IMT) of the common carotid (IMTcc), IMT of the internal carotid including plaque when present (IMTmax) and the sum of the thickest plaques present in both carotid and both common femoral bifurcations (total plaque thickness (TPT)) were measured using ultrasound. Results : People in the upper homocysteine quartile were more likely to have clinical cardiovascular disease (CVD) than those in the lowest three quartiles. They were also more likely to have plaques. The MTHFR 677C→T genotype was not associated with any of the measures of subclinical atherosclerosis in either men or women but was the most important determinant of total homocysteine levels in men under 60 years of age. Conclusions : Increased homocysteine levels but not MTHFR 677C→T genotype, are associated with subclinical atherosclerosis and the presence of plaques. Our results indicate that measurements of blood levels of homocysteine and folate in people at intermediate risk for atherosclerotic CVD before symptoms occur, might improve risk stratification and facilitate the decision to provide folate/B vitamin intervention in primary prevention. © 2009 Informa UK Ltd. All rights reserved. 13 1 11 Cited By :11

Objective: Apolipoprotein CI (apoCI) is expressed in the liver and in macrophages, and has several roles in lipid metabolism. Since macrophage apoCI expression might affect macrophage lipid homeostasis and atherosclerotic lesion development locally in the arterial wall, we investigated the effect of both systemic and macrophage apoCI on atherosclerotic lesion development. Methods and results: To investigate whether physiological expression levels of apoCI affect atherosclerosis development, we first assessed the effect of systemic endogenous apoCI expression on atherosclerosis in apoe-/-apoc1+/+ as compared to apoe-/-apoc1-/- mice at 26 weeks of age. ApoCI expression increased plasma levels of triglycerides (TG) (+70%; P < 0.01) and cholesterol (+30%; P < 0.05), and increased the atherosclerotic lesion area in the aortic root (+87%; P < 0.05). Paradoxically, incubation of apoc1+/+ and apoc1-/- murine peritoneal macrophages with AcLDL (50 μg/mL; 48 h) revealed that macrophage apoCI decreased the accumulation of cellular cholesteryl esters (CE) relatively to free cholesterol (-22%; P < 0.05). Accordingly, exogenous human apoCI increased cholesterol efflux from AcLDL-laden wild-type macrophages, and to a similar extent as apoAI and apoE. To evaluate whether atherosclerosis development would be affected by macrophage apoCI expression in vivo, we assessed atherosclerotic lesion development at 16 weeks after transplantation of bone marrow from apoe-/-apoc1-/- or apoe-/-apoc1+/+ mice to apoe-/-apoc1+/+ mice. However, in the situation wherein the liver and adipose tissue still produce apoCI, macrophage apoCI expression did not affect plasma lipid levels or the atherosclerotic lesion area. Conclusions: Systemic apoCI increases atherosclerosis, probably by inducing hyperlipidemia. Despite decreasing macrophage lipid accumulation in vitro, apoCI production by macrophages locally in the arterial wall does not affect atherosclerosis development in vivo. © 2007 Elsevier Ireland Ltd. All rights reserved.

Objective: Apolipoprotein CI (apoCI) is expressed in the liver and in macrophages, and has several roles in lipid metabolism. Since macrophage apoCI expression might affect macrophage lipid homeostasis and atherosclerotic lesion development locally in the arterial wall, we investigated the effect of both systemic and macrophage apoCI on atherosclerotic lesion development. Methods and results: To investigate whether physiological expression levels of apoCI affect atherosclerosis development, we first assessed the effect of systemic endogenous apoCI expression on atherosclerosis in apoe-/-apoc1+/+ as compared to apoe-/-apoc1-/- mice at 26 weeks of age. ApoCI expression increased plasma levels of triglycerides (TG) (+70%; P < 0.01) and cholesterol (+30%; P < 0.05), and increased the atherosclerotic lesion area in the aortic root (+87%; P < 0.05). Paradoxically, incubation of apoc1+/+ and apoc1-/- murine peritoneal macrophages with AcLDL (50 μg/mL; 48 h) revealed that macrophage apoCI decreased the accumulation of cellular cholesteryl esters (CE) relatively to free cholesterol (-22%; P < 0.05). Accordingly, exogenous human apoCI increased cholesterol efflux from AcLDL-laden wild-type macrophages, and to a similar extent as apoAI and apoE. To evaluate whether atherosclerosis development would be affected by macrophage apoCI expression in vivo, we assessed atherosclerotic lesion development at 16 weeks after transplantation of bone marrow from apoe-/-apoc1-/- or apoe-/-apoc1+/+ mice to apoe-/-apoc1+/+ mice. However, in the situation wherein the liver and adipose tissue still produce apoCI, macrophage apoCI expression did not affect plasma lipid levels or the atherosclerotic lesion area. Conclusions: Systemic apoCI increases atherosclerosis, probably by inducing hyperlipidemia. Despite decreasing macrophage lipid accumulation in vitro, apoCI production by macrophages locally in the arterial wall does not affect atherosclerosis development in vivo. © 2007 Elsevier Ireland Ltd. All rights reserved.

Background. Blood pressure, particularly pulse pressure, is associated with arterial wall stiffness, but little is known about its relation to stiffness of other parts of the body. We examined the extent to which blood pressure levels in young healthy children are related to stiffness of various tissues. Methods. In November 2000, we studied 95 healthy prepubertal children (41 boys and 54 girls, within age range 8-10 years) from two primary schools in the city of Zeist, The Netherlands. Systolic and diastolic blood pressure and pulse pressure were analyzed in relation to various tissue indicators of stiffness, including active joint mobility and skin extensibility. All results were adjusted for age, sex, body height, body weight and muscle strength as possible confounders. Results. Diastolic blood pressure was lower with increased active joint mobility (multivariate generalized linear regression coefficient = -4.5 mmHg per standard deviation [SD] joint mobility; 95% confidence interval [CI] = -7.8 to -1.2). Pulse pressure was lower with increased skin extensibility (-3.2 mmHg per SD skin extensibility; CI = -5.2 to -1.1), through a higher diastolic blood pressure (2.0 mmHg per SD skin extensibility; CI = 0.2-3.9) and possibly lower systolic blood pressure (-0.8 mmHg per SD skin extensibility; CI = -3.5 to 1.9). These associations were mutually independent. Additional adjustment for reported musculoskeletal problems or physical activity levels did not materially change the findings. Conclusions. Our findings support the hypothesis that constitutional stiffness of body tissues may be associated with blood pressure levels and eventually cardiovascular risk. Copyright © 2003 by Lippincott Williams & Wilkins, Inc.

Background. Blood pressure, particularly pulse pressure, is associated with arterial wall stiffness, but little is known about its relation to stiffness of other parts of the body. We examined the extent to which blood pressure levels in young healthy children are related to stiffness of various tissues. Methods. In November 2000, we studied 95 healthy prepubertal children (41 boys and 54 girls, within age range 8-10 years) from two primary schools in the city of Zeist, The Netherlands. Systolic and diastolic blood pressure and pulse pressure were analyzed in relation to various tissue indicators of stiffness, including active joint mobility and skin extensibility. All results were adjusted for age, sex, body height, body weight and muscle strength as possible confounders. Results. Diastolic blood pressure was lower with increased active joint mobility (multivariate generalized linear regression coefficient = -4.5 mmHg per standard deviation [SD] joint mobility; 95% confidence interval [CI] = -7.8 to -1.2). Pulse pressure was lower with increased skin extensibility (-3.2 mmHg per SD skin extensibility; CI = -5.2 to -1.1), through a higher diastolic blood pressure (2.0 mmHg per SD skin extensibility; CI = 0.2-3.9) and possibly lower systolic blood pressure (-0.8 mmHg per SD skin extensibility; CI = -3.5 to 1.9). These associations were mutually independent. Additional adjustment for reported musculoskeletal problems or physical activity levels did not materially change the findings. Conclusions. Our findings support the hypothesis that constitutional stiffness of body tissues may be associated with blood pressure levels and eventually cardiovascular risk. Copyright © 2003 by Lippincott Williams & Wilkins, Inc.