bluuebunny/crossref_metadata_2025 huggingface dataset:
Reversal of Hypercholesterolemia in Apolipoprotein E2 and Apolipoprotein E3-Leiden Transgenic Mice by Adenovirus-Mediated Gene Transfer of the VLDL Receptor
Authors: van Dijk Ko Willems , van Vlijmen Bart J. M. , van der Zee Andre , van’t Hof Belinda , van der Boom Hans , Kobayashi Kunihisa , Chan Lawrence , Havekes Louis M. , Hofker Marten H.
Published: June 2011

Abstract
—We have investigated the interaction of apolipoprotein E2(Arg
158
-Cys) (apoE2) and apolipoprotein E3Leiden (apoE3-Leiden) with the very low density lipoprotein (VLDL) receptor in vivo and in vitro to define the possible role of this receptor in lipoprotein metabolism and atherosclerosis. The in vivo binding specificity of the VLDL receptor for apoE2 and apoE3-Leiden was investigated by adenovirus-mediated gene transfer of the VLDL receptor in apoE2 and apoE3-Leiden transgenic mice lacking endogenous mouse apoE (
Apoe-/-
). Ectopic overexpression of the VLDL receptor gene in the liver resulted in a >50% decrease of plasma cholesterol levels in both apoE2 and apoE3-Leiden transgenic mice compared with liver expression of the β-galactosidase gene. This reduction in plasma cholesterol was mainly due to a reduction in the VLDL level. Overexpression of the VLDL receptor did not affect the hepatic VLDL triglyceride production, indicating that the hypocholesterolemic effect is due to an increased level of plasma clearance mediated by the VLDL receptor. In vitro binding analysis showed that both apoE2 and apoE3-Leiden VLDL compete efficiently with rabbit β-VLDL for binding to the VLDL receptor expressed on LDL receptor–deficient Chinese hamster ovary cells. We conclude from these data that both apoE2 and apoE3-Leiden function as proper ligands for the VLDL receptor in vitro and in vivo. This finding substantiates a possible role for the VLDL receptor in atherosclerosis in hyperlipidemic subjects homozygous for apoE2 or carrying apoE3-Leiden and indicates that the VLDL receptor expressed on the liver has therapeutic potential as an alternative route for clearance of binding-defective lipoproteins.
Mutations in the Low-Density Lipoprotein Receptor Gene in Chinese Familial Hypercholesterolemia Patients
Authors: Mak Ying-Tat , Pang Chi-Pui , Tomlinson Brian , Zhang Jun , Chan Yuen-Shan , Mak Tony W. L. , Masarei John R. L.
Published: June 2011

Abstract—It has been reported that in China, patients with heterozygous familial hypercholesterolemia (FH) may go unrecognized because they do not have xanthomata or premature coronary heart disease and their LDL cholesterol levels are lower than those in their Western counterparts. However, in the Chinese patients in Hong Kong, heterozygous FH appears to manifest in a way similar to that seen in Western countries or Japan. We studied sequence variations in the promoter and coding regions of the 18 exons of the LDL receptor gene in 30 Chinese FH patients. Eighteen mutations were identified in 21 patients scattered in the promoter and 10 exons. Eleven of them were first found in this study. We also found 6 polymorphisms with allelic frequencies different from those in whites but similar to the Japanese, indicating some isolation between white and Oriental populations. A total of 29 mutations in the LDL receptor gene are now known in the Chinese. There is no definite common mutation due to a founder effect. Meanwhile, there were no detectable LDL receptor gene mutations in 9 clinically diagnosed FH patients in whom the R3500Q mutation in apolipoprotein B had also been excluded. The gene defects leading to the FH phenotype in these patients may occur somewhere else in the apolipoprotein B or other related genes, or even in the noncoding sequences of the LDL receptor gene.
Concentration of Endogenous tPA Antigen in Coronary Artery Disease
Authors: Geppert Alexander , Graf Senta , Beckmann Renate , Hornykewycz Stephan , Schuster Ernst , Binder Bernd R. , Huber Kurt
Published: June 2011

Abstract
—Tissue plasminogen activator (tPA) is the major plasminogen activator responsible for dissolving blood clots found in blood vessels. However, elevated concentrations of tPA antigen were found to be related to adverse events in patients with coronary artery disease (CAD). Considerable controversy about the significance of these results exists. The goal of this cross-sectional study was to identify independent determinants for tPA antigen concentrations in patients with CAD, to possibly clarify the above paradoxical relationship. The baseline tPA antigen concentrations of 366 patients with angiographic evidence of coronary sclerosis were determined. Univariate analysis showed that age (
P
=0.013), angiographic extent of disease (
P
<0.001), presence of angina at rest (
P
<0.001), diabetes mellitus (
P
=0.004), hypercholesterolemia (
P
=0.045), hypertriglyceridemia (
P
=0.015), and chronic intake of nitrates (
P
<0.001) were significantly and positively related to tPA antigen concentration, while the chronic intake of aspirin was inversely related to tPA antigen (
P
<0.001). In addition, plasminogen activator inhibitor type 1 (PAI-1) activity was found to be significantly and positively associated with tPA antigen concentration (
P
<0.001). A multivariate analysis identified chronic low-dose aspirin therapy (
P
<0.001), PAI-1 activity (
P
<0.001), hypertriglyceridemia (
P
=0.005), the type of angina (
P
=0.026), multivessel disease (
P
=0.041), and hypercholesterolemia (
P
=0.043) as significant and independent determinants of tPA antigen. While hypertriglyceridemia and hypercholesterolemia both are related to the underlying disease, the type of angina and the number of involved vessels are linked to the severity and extent of disease, and all of them are indicators of a prothrombotic state found during the progression of CAD. In contrary, low-dose aspirin rather would decrease the likelihood of thrombotic events. The relation of tPA antigen to PAI-1 activity furthermore underlines the relation between tPA antigen concentration and a prothrombotic state. Therefore, the positive or—in case of aspirin therapy—negative correlation of these parameters with tPA antigen concentration would indicate that thrombus formation and simultaneous endothelial cell activation might be major determinants for tPA antigen concentration in CAD.
Activation of Matrix-Degrading Metalloproteinases by Mast Cell Proteases in Atherosclerotic Plaques
Authors: Johnson Jason L. , Jackson Christopher L. , Angelini Gianni D. , George Sarah J.
Published: June 2011

Abstract—Recent studies suggest that mast cell–derived neutral proteases can activate matrix-degrading metalloproteinases (MMPs). We have investigated the role of the mast cell proteases tryptase and chymase in the activation of MMPs in human carotid endarterectomy specimens (atherosclerotic, n=32) and postmortem carotid arteries (control, n=17). In vitro degranulation of mast cells in atherosclerotic carotid arteries by compound 48/80 caused a significant increase in MMP activity. Addition of the nonselective tryptase inhibitor antipain, the specific trypsinlike protease inhibitor 4-amidinophenylmethanesulfonyl fluoride, and the chymase inhibitor chymostatin reduced this increase in MMP activity by 30±6%, 23±6%, and 9±2%, respectively. Immunocytochemistry identified significantly higher numbers of tryptase-containing cells (mast cells) and cells expressing MMP-1 and MMP-3 in the “shoulder” regions of atherosclerotic artery lesions compared with the tunica media of control arteries. Dual immunocytochemistry showed collocation of MMP-1 and MMP-3 with mast cells in the shoulder regions. Degranulation was observed in 78±5% (mean±SEM) of mast cells in this area, whereas nonactivated mast cells were observed in all other areas. In situ zymography revealed caseinolytic and gelatinolytic activity in these areas. In conclusion, in vitro mast cell degranulation, which releases mast cell proteases, in carotid arteries increases MMP activity. Furthermore, elevated MMP-1 and MMP-3 expression is collocated with increased numbers of degranulated mast cells and with greater MMP activity in the shoulder regions of atherosclerotic plaques. Activation of MMPs by mast cell–derived proteases may be an important mechanism in atherosclerotic plaque destabilization.
Frequency of ApoB and ApoE Gene Mutations as Causes of Hypobetalipoproteinemia in the Framingham Offspring Population
Authors: Welty Francine K. , Lahoz Carlos , Tucker Katherine L. , Ordovas Jose M. , Wilson Peter W. F. , Schaefer Ernst J.
Published: June 2011

Abstract
—Hypobetalipoproteinemia (HBLP) is characterized by plasma concentrations of apolipoprotein B (apoB) and low density lipoprotein cholesterol (LDL-C) below the fifth percentile. Some forms of HBLP have been shown to be due to truncated forms of apoB-100. A total of 3873 subjects participating in the Framingham Offspring Study had LDL-C levels measured every 4 to 5 years throughout a 25-year period. Seventy-five subjects were identified with persistent HBLP, defined as an LDL-C <70 mg/dL on at least 2 observations, for a prevalence of 1.9% in this population. Compared with subjects with LDL- C ≥70 mg/dL, subjects with HBLP had significantly lower mean levels of total cholesterol, LDL-C, triglyceride, and apoB; higher levels of high density lipoprotein cholesterol; and a higher prevalence of the E2/E3 genotype: 38.7% versus 10.9% (
P
<0.001). Men with HBLP had a larger mean LDL particle size than did men with an LDL- C ≥70 mg/dL. One individual had a truncated apoB as a cause of HBLP, for a prevalence of 0.03%. Medical causes of HBLP included 2 cases of Crohn’s disease, 1 of hemochromatosis, and 1 of hepatitis. Three subjects with HBLP developed coronary heart disease, for an incidence of 4% compared with 5% in those with an LDL- C ≥70 mg/dL (
P
=NS). The incidence of cancer was 8% in those with HBLP compared with 4% in those with an LDL-C ≥70 mg/dL (
P
=0.21). In conclusion, a truncated apoB was a rare cause of HBLP, whereas the E2/E3 genotype was a much more common cause. A large prospective study is needed to evaluate the incidence of cancer and atherosclerosis in subjects with HBLP.
Circulating Cell Adhesion Molecules Are Correlated With Ultrasound-Based Assessment of Carotid Atherosclerosis
Authors: Rohde Luis E. , Lee Richard T. , Rivero Jose , Jamacochian Marika , Arroyo Luis H. , Briggs Willian , Rifai Nader , Libby Peter , Creager Mark A. , Ridker Paul M.
Published: June 2011

Abstract
—Although cellular adhesion molecules (CAMs) are hypothesized to play an important role in atherogenesis, the relationship between CAMs and systemic atherosclerosis is uncertain. Among 92 outpatients (48 men; mean±SD age, 65±9 years), we evaluated the association of soluble vascular CAM-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) with carotid intimal-medial thickness (IMT), an index of early atherosclerosis. All subjects underwent a 2-dimensional ultrasound examination of both carotid arteries at the distal common carotid arteries and bifurcation. sVCAM-1 and sICAM-1 levels measured by enzyme-linked immunosorbent assay were significantly correlated with mean IMT of the common carotid artery (
r
=0.34 and
r
=0.30, respectively;
P
<0.01) and carotid bifurcation (
r
=0.31 and
r
=0.26, respectively;
P
<0.05), whereas sVCAM-1 was also positively associated with maximal carotid IMT (
r
=0.35,
P
<0.01). Adjustment for age attenuated the association between sVCAM-1 and common (
r
=0.16,
P
=0.13) and bifurcation (
r
=0.18,
P
=0.07) carotid IMT but had minimal effect on the associations between sICAM-1 and carotid measurements (
r
=0.32,
P
<0.01;
r
=0.23,
P
<0.05; for common and bifurcation IMT, respectively). Age-adjusted sICAM-1 levels increased in a stepwise fashion across common carotid IMT tertiles (253±27 versus 275±24 versus 384±26 pg/mL for the lowest, intermediate, and highest IMT tertiles, respectively;
P
<0.01). A similar trend was also found between sVCAM-1 levels and common carotid IMT tertiles (625±60 versus 650±53 versus 714±58 pg/mL;
P
<0.15). These associations were minimally affected in analyses adjusting for hypertension, diabetes, smoking, low and high density lipoprotein cholesterol, lipoprotein(a), and homocysteine, or in a subgroup analysis limited to those with no prior history of atherothrombotic disease. These data demonstrate a positive association between serum CAMs with carotid IMT and further support the hypothesis that systemic inflammation may have a role in atherosclerotic lesion development.
Genotype-Specific Transcriptional Regulation of PAI-1 Gene by Insulin, Hypertriglyceridemic VLDL, and Lp(a) in Transfected, Cultured Human Endothelial Cells
Authors: Grenett Hernan E. , Benza Raymond L. , Fless Gunther M. , Li Xin-Nong , Davis Glenda C. , Booyse Francois M.
Published: June 2011

Abstract
—Plasminogen activator inhibitor-1 (PAI-1) has been shown to be an independent risk factor for coronary artery disease. Variations in plasma PAI-1 levels have been attributed to variations in the PAI-1 gene, and associations between PAI-1 levels and PAI-1 genotypes suggest that PAI-1 expression may be regulated in a genotype-specific manner by insulin, hypertriglyceridemic (HTG) very low density lipoprotein (VLDL), or lipoprotein(a) [Lp(a)]. Polymerase chain reaction–amplified 1106-bp fragments of the promoter of the 1/1 and 2/2 PAI-1 genotypes were sequenced and showed 5 regions of small nucleotide differences in the 1/1 versus 2/2 PAI-1 promoters that consistently occurred with high frequency. These fragments were ligated into the luciferase reporter gene, and 1/1 and 2/2 PAI-1 genotype human umbilical vein endothelial cell (HUVEC) cultures were transiently transfected with their respective p1PAI110/luc and p2PAI110/luc constructs and vice versa. Insulin induced an ≈12- to 16-fold increase in luciferase activity in both the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with the p1PAI110/luc construct. HTG-VLDL and Lp(a) induced luciferase activity by ≈14- to 16- and ≈8- to 11-fold, respectively, in both the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with the p2PAI110/luc construct. The positive control interleukin-1 showed an ≈7- to 12-fold response in the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with either of the constructs. These cross-over results demonstrate that regulation of either the 1/1 or 2/2 PAI-1 genotype by its respective inducer is due to the promoter itself and not to some factor(s) expressed differently in the 1/1 or 2/2 PAI-1 genotype HUVEC cultures.
Expression of Thrombomodulin in Atherosclerotic Lesions and Mitogenic Activity of Recombinant Thrombomodulin in Vascular Smooth Muscle Cells
Authors: Tohda Gen , Oida Koji , Okada Yoshikatsu , Kosaka Shotaro , Okada Eiko , Takahashi Sadao , Ishii Hidemi , Miyamori Isamu
Published: June 2011

Abstract
—Thrombomodulin (TM), a thrombin receptor protein found on the endothelial cell surface, contains 6 tandem epidermal growth factor (EGF)–like structures. Recombinant human TM peptide containing these 6 EGF-like domains (rTM
e
1–6) exhibits mitogenic activity in Swiss 3T3 cells. We examined the localization of TM in atherosclerotic lesions and the effects of rTM
e
1–6 on the growth of cultured rat vascular smooth muscle cells (SMCs). Immunohistochemical analysis demonstrated that TM antigen was localized on monocytes, macrophages, and vascular SMCs. In cultured vascular SMCs, rTM
e
1–6 accelerated [
3
H]thymidine uptake into DNA in a dose-dependent manner up to 3.4 times the control level. This mitogenic activity was abolished by addition of polyclonal anti-human TM antibody. The rTM
e
1–6–induced mitogenesis was enhanced by EGF. However, a neutralizing monoclonal antibody against the EGF receptor (monoclonal antibody 225) did not inhibit the mitogenic activity of rTM
e
1–6. Calphostin C, a specific protein kinase C inhibitor, and lavendustin-A, an inhibitor of EGF receptor–specific protein tyrosine kinase, inhibited the mitogenic activities of both rTM
e
1–6 and EGF. Finally, rTM
e
1–6 treatment increased the level of phosphorylated mitogen-activated protein kinase in SMCs. Together, these results suggest that TM expression in atherosclerotic lesions may be associated with promotion of atherosclerosis through its mitogenic activity in vascular SMCs.
Upregulation of Prostacyclin Synthesis–Related Gene Expression by Shear Stress in Vascular Endothelial Cells
Authors: Okahara Kazuhiro , Sun Bing , Kambayashi Jun-ichi
Published: June 2011

Abstract
—Prostacyclin (prostaglandin I
2
, PGI
2
) has a variety of functions, including inhibition of smooth muscle cell proliferation, vasodilation, and antiplatelet aggregation. PGI
2
production in endothelial cells has been reported to increase biphasically after shear loading, but the underlying mechanism is not well understood. To clarify the mechanism for the second phase of PGI
2
upregulation, we examined the gene expression of the enzymes involved in PGI
2
production in human umbilical vein endothelial cells (HUVECs) after shear-stress (24 dyne/cm
2
) loading. The production of 6-keto-PGF
1α
, a stable metabolite of PGI
2
, increased time-dependently under shear stress. The arachidonic acid liberation from membrane phospholipids in HUVECs after 12 hours of shear loading was increased significantly compared with the static condition. No change was observed for cytosolic phospholipase A
2
expression, as detected by reverse transcription–polymerase chain reaction and Western blotting. Cyclooxygenase (COX)-1 mRNA increased after 1 hour of shear loading, and the increase lasted for 12 hours, the longest time tested, whereas COX-2 mRNA increased after 1 hour of shear loading and peaked at 6 hours. An increase of COX-1 expression was detected at 12 hours of shear loading by Western blotting. No expression of COX-2 was detected in the static control, but induced expression was observed at 6 hours after shear loading. PGI
2
synthase was also found to be upregulated. These results suggest that the elevated PGI
2
production by shear stress is mediated by increased arachidonic acid release and a combination of increased expression of COXs and PGI
2
synthase.
Effect of the Novel Antiplatelet Agent Cilostazol on Plasma Lipoproteins in Patients With Intermittent Claudication
Authors: Elam M. B. , Heckman J. , Crouse J. R. , Hunninghake D. B. , Herd J. A. , Davidson M. , Gordon I. L. , Bortey E. B. , Forbes W. P.
Published: June 2011

Abstract
—Cilostazol is an antiplatelet agent and vasodilator marketed in Japan for treatment of ischemic symptoms of peripheral vascular disease. It is currently being evaluated in the United States for treatment of symptomatic intermittent claudication (IC). Cilostazol has been shown to improve walking distance in patients with IC. In addition to its reported vasodilator and antiplatelet effects, cilostazol has been proposed to have beneficial effects on plasma lipoproteins. We examined the effect of cilostazol versus placebo on plasma lipoproteins in 189 patients with IC. After 12 weeks of therapy with 100 mg cilostazol BID, plasma triglycerides decreased 15% (
P
<0.001). Cilostazol also increased plasma high density lipoprotein cholesterol (HDL-C) (10%) and apolipoprotein (apo) A1 (5.7%) significantly (
P
<0.001 and
P
<0.01, respectively). Both HDL
3
and HDL
2
subfractions were increased by cilostazol; however, the greatest percentage increase was observed in HDL
2
. Individuals with baseline hypertriglyceridemia (>140 mg/dL) experienced the greatest changes in both HDL-C and triglycerides with cilostazol treatment. In that subset of patients, HDL-C was increased 12.2% and triglycerides were decreased 23%. With cilostazol, there was a trend (3%) toward decreased apoB as well as increased apoA1, resulting in a significant (9.8%,
P
<0.002) increase in the apoA1 to apoB ratio. Low density lipoprotein cholesterol and lipoprotein(a) concentrations were unaffected. Cilostazol treatment resulted in a 35% increase in treadmill walking time (
P
=0.0015) and a 9.03% increase in ankle-brachial index (
P
<0.001). These results indicate that in addition to improving the symptoms of IC, cilostazol also favorably modifies plasma lipoproteins in patients with peripheral arterial disease. The mechanism of this effect is currently unknown.
Contrasting Patterns of Spontaneous Aortic Disease in Young and Old Rabbits
Authors: Barnes Sally E. , Weinberg Peter D.
Published: June 2011

Abstract
—The pattern of spontaneous lipid deposition around aortic branch ostia was mapped in New Zealand White rabbits aged 1 month or 2 to 6 years. The young rabbits were studied within 1 day of weaning, and the older rabbits had been maintained on a low-protein, low-fat diet. Plasma concentrations of total cholesterol for the two groups averaged 75 and 18 mg/dL, respectively. Aortas were fixed in situ at a pressure of 90 to 100 cm H
2
O, stained with oil red O, and photographed en face under an epifluorescence microscope. Areas of staining contrasted in color with the fixative-stimulated autofluorescence of nondiseased tissue and were mapped by placing grids over the photomicrographs. Disease around intercostal ostia was rare, but two distributions were established by combining data from many branches. In weanlings, staining was seen within a triangular area downstream of the branch. In old animals, this area had the lowest frequency of disease; lesions tended to occur downstream of the spared region, along axes lying to either side of it, and at the lateral and upstream margins of the ostium. Disease was less rare at celiac branches. It occurred mainly downstream of the ostium in weanlings, whereas upstream sites were most affected in old animals, although significant disease remained at the juvenile locations. Earlier reports have described similar age-related distributions of disease in human aortas, consistent with a common underlying mechanism. The distributions also correlate with the spatial variations in arterial transport properties established in previous studies, and may be determined by them.
Similar Effects of Diets Rich in Stearic Acid or
trans
-Fatty Acids on Platelet Function and Endothelial Prostacyclin Production in Humans
Authors: Turpeinen Anu M. , Wübert Joachim , Aro Antti , Lorenz Reinhard , Mutanen Marja
Published: June 2011

Abstract
—The effects of stearic acid (C18:0) and
trans-
fatty acids (
trans-
FAs) on measures of platelet function and prostacyclin (PGI
2
) production are poorly understood in humans. In this controlled dietary study, platelet function and endothelial PGI
2
production were studied in healthy humans after they consumed diets rich in C18:0 or
trans-
FAs. For 5 weeks, 80 subjects consumed a baseline diet high in saturated FAs and were then switched to a diet containing 9.3% of energy as stearic acid or a diet containing 8.7 energy% as
trans-
FAs from hydrogenated vegetable oils for another 5 weeks. All diets contained 32.2 to 33.9 energy% fat, 14.6 to 15.8 energy% saturated plus
trans-
FAs, 12.2 to 12.5 energy%
cis
-monounsaturated, and 2.9 to 3.5 energy% polyunsaturated FAs. No significant differences between the C18:0 and
trans-
FA diets were found in the urinary excretion of 2,3-dinor-thromboxane B
2
or 2,3-dinor-6-keto-prostaglandin F
1α
. In vitro production of thromboxane B
2
by platelets as well as urinary excretion of β-thromboglobulin were also similar after both diets. Collagen-induced in vitro aggregation was significantly enhanced after the C18:0 diet compared with the
trans-
FA diet (
P
=.02), whereas no differences between the diets were found with ADP. The results indicate similar effects of C18:0 and
trans-
FA diets on platelet activation and endothelial PGI
2
production.
Smooth Muscle Cell Heterogeneity
Authors: Shanahan Catherine M. , Weissberg Peter L.
Published: June 2011

Abstract
—Early morphological and biochemical studies indicated that vascular smooth muscle cells (VSMCs) exhibited two distinct phenotypes and that a change from the contractile to the synthetic phenotype was a prerequisite for progression of vascular disease. More recently, it has become evident that these phenotypes probably represent the extremes of a spectrum of phenotypes that may coexist in the vessel wall, which are dictated by their environment and functional requirements and which reflect differing patterns of gene expression. Therefore, knowledge of the key factors that regulate these patterns of gene expression is likely to lead to the ability to manipulate VSMC phenotype. However, before such factors can be identified, the relationship between VSMC gene expression and VSMC phenotype must first be established. We therefore undertook a differential screen of cDNA from VSMCs in vitro to provide a bank of gene markers that could be used under a variety of circumstances to define VSMC phenotype in terms of the pattern of genes expressed. Using this approach, we have found that the pattern of gene expression that occurs during neointima formation in the balloon-injured rat carotid artery is very similar to that seen at a specific period in the developing aorta of the early neonate and is characterized by coexpression of genes for both contractile and matrix proteins. Furthermore, recent studies have shown that VSMCs isolated at different stages of aortic development can stably maintain different phenotypic characteristics in cell culture. The use of these cells in transfection experiments with SM-specific promoter–chloramphenicol acetyltransferase reporter constructs may enable us to determine what regulates the pattern of gene expression in different VSMC phenotypes. Such studies may ultimately lead to the identification of transcription factors responsible for determining VSMC phenotype and may therefore provide targets for therapy aimed at manipulating VSMC gene expression in vascular disease.
Herpesviruses in Atherosclerosis and Thrombosis
Authors: Nicholson Andrew C. , Hajjar David P.
Published: June 2011

Abstract—The role of herpesvirus infections in the pathogenesis of vascular diseases remains an enigma. Although there is abundant circumstantial evidence of a role for herpesviruses in atherosclerosis and related processes, a cause-and-effect relationship has yet to be definitively established. This article will review the pathological, molecular, and biochemical evidence supporting the hypothesis that herpesviruses are involved in the development of atherosclerosis, restenosis after coronary angioplasty, accelerated atherosclerosis in recipients of heart transplants, and the induction of a prothrombotic phenotype in vascular endothelial cells.
Effects of Intracellular Free Cholesterol Accumulation on Macrophage Viability
Authors: Kellner-Weibel G. , Jerome W. G. , Small D. M. , Warner G. J. , Stoltenborg J. K. , Kearney M. A. , Corjay M. H. , Phillips M. C. , Rothblat G. H.
Published: June 2011

Abstract
—This study was designed to identify cellular responses associated with free cholesterol (FC) accumulation in model macrophage foam cells. Mouse peritoneal macrophages (MPMs) or J774 macrophages were loaded with cholesteryl esters using acetylated LDL and FC/phospholipid dispersions and were subsequently exposed to an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. This treatment produced a rapid accumulation of cellular FC. The FC that accumulated due to ACAT inhibition was more readily available for efflux to 2-hydroxypropyl-β-cyclodextrin (which removes cholesterol from the plasma membrane) than FC in untreated control cells. After a 3-hour exposure to an ACAT inhibitor, a significant increase in phospholipid synthesis was seen, followed by the leakage of LDH after 12 hours of treatment. We also observed, by electron and fluorescence microscopy, morphological indications of both apoptosis and necrosis in cells treated with an ACAT inhibitor. In addition, inhibition of ACAT for 48 hours resulted in the formation of FC crystals in MPMs but not in J774 cells. If compound 3β-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A), which modulates intracellular trafficking of cholesterol, was added together with the ACAT inhibitor, each of the metabolic changes elicited by the accumulation of excess FC was either diminished or eliminated. The protective affect of U18666A was not due to a decrease in cellular FC concentrations, because cells treated with an ACAT inhibitor accumulated similar amounts of FC in the presence or absence of U18666A. Thus, treatment with U18666A results in the sequestering of FC in a pool that prevents it from causing various responses to FC deposition in macrophages. The metabolic changes that were produced when these model foam cells were treated with the ACAT inhibitor parallel the pathological events that have been shown to occur in the developing atherosclerotic plaque.
Possible Involvement of m-Calpain in Vascular Smooth Muscle Cell Proliferation
Authors: Ariyoshi Hideo , Okahara Kazuhiro , Sakon Masato , Kambayashi Jun-ichi , Kawashima Sei-ichi , Kawasaki Tomio , Monden Morito
Published: June 2011

Abstract
—Vascular smooth muscle cell (VSMC) proliferation still remains a poorly understood process, although it is believed to play a critical role in pathological states, including atherosclerosis and hypertension. Several reports have suggested that proteases may be directly involved in this process; however, it was still unclear which protease is responsible for VSMC proliferation. In this study, by use of a cell-permeable calpain inhibitor (calpeptin; benzyloxycarbonyl-Leu-nLeu-H), its analogue (benzyloxycarbonyl-Leu-Met-H), the cell-impermeable serine protease inhibitor leupeptin, and antisense oligonucleotide against m-calpain to inhibit proliferation of primarily cultured human VSMCs, we investigated whether calcium-activated neutral protease (calpain) is involved in VSMC proliferation. Calpeptin and its analogue, more specific for m-calpain, equally inhibited the proliferation of VSMCs in a dose-related manner, whereas a more limited antiproliferative effect was observed in leupeptin-treated VSMCs. Antisense oligonucleotide against m-calpain, but not scrambled antisense, dose-dependently inhibited m-calpain expression and proliferation of VSMCs. Maximal inhibition was an ≈50% reduction of cell number and m-calpain antigen observed at 50 μmol/L of antisense oligonucleotide. Calpeptin or antisense oligonucleotide against m-calpain increased the expression of the endogenous calpain substrate pp125FAK (focal adhesion kinase), whereas the expression of the endogenous calpain inhibitor calpastatin was not affected. These results suggest that the proliferation of VSMCs requires protease activity, some of which is due to m-calpain.
Ultrastructural Localization of Secretory Type II Phospholipase A
2
in Atherosclerotic and Nonatherosclerotic Regions of Human Arteries
Authors: Romano Mirtha , Romano Egidio , Björkerud Sören , Hurt-Camejo Eva
Published: June 2011

Abstract
—We recently reported on the immunolocalization of type II secretory nonpancreatic phospholipase A
2
(snpPLA
2
) in human atherosclerotic lesions. In the present study, we present data on the distribution and ultrastructural localization of snpPLA
2
in adjacent nonatherosclerotic and atherosclerotic regions of human arteries. Electron microscopy (EM) of immunogold labeling techniques with a monoclonal antibody was used to analyze arterial tissue. The human specimens analyzed were obtained from autopsy and surgery cases. The results with EM showed a stronger snpPLA
2
immunoreactivity in regions of arteries with atherosclerotic lesions than in regions without lesions from the same individual. snpPLA
2
immunoreactivity was stronger in the arterial intima of atherosclerotic than of nonatherosclerotic tissue. EM-immunogold examination revealed that the majority of snpPLA
2
was localized along the extracellular matrix, associated with collagen fibers and other extracellular matrix structures. Intracellular snpPLA
2
was observed in electron-dense vesicles in intimal cells. snpPLA
2
was also found in contact with large, extracellular lipid droplets. These results support the hypothesis that extracellular snpPLA
2
is localized at sites where it may hydrolyze phospholipids from lipoproteins and lipid aggregates retained in the extracellular matrix of the arterial wall. This may be a mechanism for in situ release of proinflammatory lipids, free fatty acids, and lysophosphatidylcholine in regions of apolipoprotein B accumulation, which are abundant in atherosclerotic lesions.
Distribution and Synthesis of Apolipoprotein J in the Atherosclerotic Aorta
Authors: Ishikawa Yukio , Akasaka Yoshikiyo , Ishii Toshiharu , Komiyama Kazuo , Masuda Shigeru , Asuwa Noriko , Choi-Miura Nam-Ho , Tomita Motowo
Published: June 2011

Abstract—
The distribution of apolipoprotein (apo) J during the development of atherosclerosis in the human aorta was evaluated by immununohistochemical observation, together with the other apolipoprotein A-I, A-II, B, C-III, and E. Although apoJ was never observed in the normal aorta (ie, without any intimal lesions or intimal thickening), it was distributed not only in the intima but also in the media of aortas with diffuse, intimal thickening or atherosclerotic lesions. Double immunostaining with antibodies for apoJ and α-smooth muscle actin revealed apoJ deposition in smooth muscle cells (SMCs) or the aortic stroma in the vicinity of SMCs. The extent of apoJ distribution in the aortic wall increased with the degree of atherosclerosis development. In addition, the distribution pattern of apoJ was very similar to that of apoA-I and E. In situ hybridization with human apoJ cDNA demonstrated intense signals in cells scattered within the subendothelial space and medial SMCs of the aorta with advanced atherosclerosis but not in those of the normal aorta without intimal thickening. Furthermore, reverse transcriptase–polymerase chain reaction of the cultured human aortic SMCs revealed apoJ mRNA expression in these cells. The results indicate that apoJ in the aortic wall originates from not only apoJ circulated in the plasma but also apoJ produced by SMCs in the aortic wall. Considering the similarities of the distribution between apoJ and apo-A-I or E, we hypothesize that apoJ possibly has a protective role against human atherosclerosis by its involvement with cholesterol transport from the aortic wall to the liver.
The Relation Between De Novo Atherosclerosis Remodeling and Angioplasty-Induced Remodeling in an Atherosclerotic Yucatan Micropig Model
Authors: de Smet Bart J. G. L. , Pasterkamp Gerard , van der Helm Yvonne J. , Borst Cornelius , Post Mark J.
Published: June 2011

Abstract
—Geometric remodeling in de novo atherosclerosis and in restenosis after balloon angioplasty constitutes a change in total arterial circumference that, together with plaque growth or neointima formation, determines the lumen of the artery. The heterogeneous nature of arterial obstructions raises the question of whether early and late outcomes (restenosis) of angioplasty are affected by the degree and direction of de novo atherosclerotic remodeling. This study was designed to assess the relationship between atherosclerotic remodeling and the degree and mechanism of restenosis after balloon angioplasty. Atherosclerosis was induced in 27 peripheral arteries of 18 Yucatan micropigs by a combination of denudation and atherogenic diet. Balloon angioplasty was performed, with serial intravascular ultrasound and quantitative angiography before and after intervention and at 42 days’ follow-up. We used the relative media-bounded area (MBA), defined as the MBA of the treated site divided by the MBA of the reference, before angioplasty as a measure of remodeling in de novo atherosclerosis and late MBA loss as a measure of remodeling after balloon angioplasty. Relative MBA before angioplasty was not correlated with angiographic and echographic acute gain after balloon angioplasty (
r=
.22,
P
=.28 and
r=
.14,
P
=.48) or with late lumen loss (
r=
−.05,
P
=.81 and
r=
.19,
P
=.33). No correlation was found between relative MBA and late MBA loss (
r=
.14 and
P
=.48). In the atherosclerotic Yucatan micropig, remodeling during de novo atherosclerosis has no relevance for acute gain and late lumen loss after balloon angioplasty. Both the direction and the extent of remodeling after balloon angioplasty are not related to the direction and extent of remodeling during de novo atherosclerosis.
Pretreatment of Rabbits With Either Hirudin, Ancrod, or Warfarin Significantly Reduces the Immediate Uptake of Fibrinogen and Platelets by the Deendothelialized Aorta Wall After Balloon-Catheter Injury In Vivo
Authors: Hatton Mark W. C. , Ross Bonnie , Southward Suzanne M. R. , DeReske Marnie , Richardson Mary
Published: June 2011

Abstract
—Fibrinogen and platelets rapidly saturate the exposed subendothelium of a freshly deendothelialized aorta in vivo. As thrombin generated within the site of injury is largely responsible for fibrin(ogen) deposition, we questioned whether various anticoagulant treatments would inhibit uptake of both fibrinogen and platelets in vivo. Rabbits were anticoagulated by pretreatment with either Warfarin, Ancrod, or recombinant hirudin. Each anesthetized, anticoagulated (or saline-injected control) rabbit was injected IV with rabbit
51
Cr-platelets and
125
I-fibrinogen before a balloon-catheter deendothelializing (or sham) injury of the thoracic aorta. At 10 minutes after injury, the rabbit was exsanguinated and the aorta excised. Platelet adsorption by the deendothelialized aorta surface was substantially reduced in anticoagulated rabbits (controls, 2.2×10
5
/mm
2
; Warfarin-treated, 1.2×10
5
/mm
2
; Ancrod-treated, 5.3×10
4
/mm
2
; r-hirudin–treated [5 mg/kg], 5.3×10
4
/mm
2
), and a significant reduction of fibrinogen associated with the platelet layer (from 5.3 to 1 to 2 pmol/cm
2
) and within the underlying intima-media layer (from 16.9 to 5 to 6 pmol/cm
2
) was observed in the r-hirudin–and Warfarin-treated rabbits. The pattern of aorta-deposited
51
Cr-platelets and
125
I-fibrin in the anticoagulated rabbits corresponded well with an assessment by transmission electron microscopy of aortic tissue samples. We conclude that ≈70% of fibrinogen uptake is thrombin dependent and that ≈80% of platelet adsorption depends on codeposited fibrin(ogen) during the 10-minute interval after balloon injury. Pretreatment with an agent that interferes with either thrombin or fibrin production will inhibit the immediate interaction of fibrinogen and platelets with the freshly exposed subendothelium.
Development of Compartmental Models in Stable-Isotope Experiments
Authors: Pont Frédéric , Duvillard Laurence , Vergès Bruno , Gambert Philippe
Published: June 2011

Abstract
—Kinetic experiments are of great importance in lipid research because they further the understanding of lipid metabolism in vivo and help to explain the physiopathology of lipid disorders in humans. At present, due to species specificity, no valid animal model can efficiently replace a study in humans to explore lipid metabolism, and the use of radioactive tracers is restricted in humans. Thus, stable-isotope tracer kinetic studies have become an important component of research programs to achieve in humans a quantitative understanding of the dynamics of metabolic processes in vivo. The aim of this review is to describe the practical aspects of compartmental model development in stable-isotope experiments. The recent development of computer hardware and modeling software has dramatically facilitated the task of the modeler in his or her calculations. In the current review, we show that the model may be considered an integral component of the experimental design and that model development must obey strict rules to provide a rigorous solution. The main difficulties of model development in tracer experiments, such as experiment design, model identifiability, data expression, comparison of models, or tracer recycling, are presented with extensive references. We have paid particular attention to kinetic modeling in stable-isotope experiments because they have shown the greatest development in recent years.
Inhibition of Repetitive Thrombus Formation in the Stenosed Canine Coronary Artery by Enoxaparin, But Not by Unfractionated Heparin
Authors: Leadley Robert J. , Kasiewski Charles J. , Bostwick Jeffrey S. , Bentley Ross , Dunwiddie Christopher T. , Perrone Mark H.
Published: June 2011

Abstract
—Experiments were designed to compare the antithrombotic efficacy of enoxaparin and unfractionated heparin (UH) in a model of platelet-dependent cyclic flow reductions (CFRs) in the stenosed canine circumflex coronary artery. Low-molecular-weight heparins (LMWHs) are safe and effective in the prevention and treatment of venous thromboembolism. The present experiments were designed to evaluate the potential use of LMWHs in arterial thrombotic indications by comparing the antithrombotic effect of an LMWH with that of UH in an animal model of unstable angina. After establishment of consistent CFRs by experimentally induced vascular stenosis and damage, vehicle (saline), enoxaparin, or UH was administered intravenously as a loading dose plus a continuous infusion for 1 hour. The inhibition of CFRs was taken as an indicator of antithrombotic efficacy. Enoxaparin inhibited repetitive platelet thrombus formation in a dose-dependent manner, with significant inhibition of CFRs achieved at 0.5 mg/kg+5 μg/kg per minute. This dose of enoxaparin resulted in anti-Xa levels of 0.9 to 1.0 IU/mL, anti-IIa levels of 0.2 to 0.3 IU/mL, activated partial thromboplastin time (APTT) of 1.3-fold over baseline, and a 1.4-fold increase (NS) in template bleeding time. Near-complete abolishment of CFRs was achieved with enoxaparin at 1.0 mg/kg+10 μg/kg per minute. This dose of enoxaparin produced anti-Xa levels of 2 to 2.2 IU/mL, anti-IIa levels of 0.5 to 0.6 IU/mL, an increase in APTT of 1.4- to 1.5-fold over baseline, and a 1.9-fold increase (
P
<0.05) in template bleeding time. In contrast, UH had no significant effect on CFRs at a dose (100 U/kg+10 U/kg per minute) that resulted in anti-Xa levels of 1.2 to 1.6 IU/mL, anti-IIa levels of 1.8 to 2.4 IU/mL, an increase in APTT greater than 10-fold over baseline, and a 2.5-fold increase (
P
<0.05) in template bleeding time. Compared with the vehicle group, circulating platelet count and hematocrit were not changed significantly by any dose of enoxaparin or UH tested. Enoxaparin, unlike UH, prevented repetitive platelet-dependent thrombus formation in the dog, thereby supporting the potential use of enoxaparin as a replacement for heparin in the treatment of arterial thrombotic disorders such as unstable angina.
Hyperleptinemia as a Component of a Metabolic Syndrome of Cardiovascular Risk
Authors: Leyva Francisco , Godsland Ian F. , Ghatei Mohammed , Proudler Anthony J. , Aldis Stephen , Walton Christopher , Bloom Stephen , Stevenson John C.
Published: June 2011

Abstract
—In humans, production of the adipocyte-derived peptide leptin has been linked to adiposity, insulin, and insulin sensitivity. We therefore considered that alterations in plasma leptin concentrations could constitute an additional component of a metabolic syndrome of cardiovascular risk. To explore this hypothesis, we employed factor analysis, a multivariate statistical technique that allows reduction of large numbers of highly intercorrelated variables to composite, biologically meaningful factors. Seventy-four men [age, 48.4±1.3 years (mean±SEM); body mass index (BMI), 25.6±0.3 kg/m
2
] who were free of coronary heart disease and diabetes underwent anthropometric measurements (subscapular-to-triceps [S:T] and subscapular-to-biceps [S:B] skinfold thickness ratios, measurement of fasting plasma leptin, and an intravenous glucose tolerance test (IVGTT) for assessment of insulin sensitivity. Plasma leptin concentrations were correlated with BMI (
r
=0.57,
P
<0.001), S:T (
r
=0.34,
P
=0.003), S:B (
r
=0.37,
P
<0.001), systolic and diastolic blood pressures (both
r
=0.24,
P
=0.044), fasting triglycerides (
r
=0.31,
P
=0.007), serum uric acid (
r
=0.35,
P
=0.003), fasting glucose (
r
=0.32,
P
=0.003) and insulin (
r
=0.33,
P
=0.004), and IVGTT insulin (
r
=0.63,
P
<0.001). A negative correlation was observed between leptin and insulin sensitivity (
r
=−0.32,
P
=0.006). No significant correlations emerged between plasma leptin concentrations and age, high density lipoprotein cholesterol, or IVGTT glucose. In multivariate regression analyses, BMI (standardized coefficient [SC] =0.40,
P
=0.001), fasting insulin (SC=0.23,
P
=0.036), and IVGTT insulin (SC=0.51,
P
<0.001) emerged as independent predictors of plasma leptin concentrations (
R
2
=0.56,
P
<0.001). After adjustment for BMI, only IVGTT insulin emerged as a significant predictor of plasma leptin concentrations (SC=0.56,
P
<0.001,
R
2
=0.45,
P
<0.001). Factor analysis of plasma leptin concentrations and the variables that are considered relevant to the insulin resistance syndrome revealed a clustering of plasma leptin concentrations with a factor dominated by insulin resistance and high IVGTT insulin, separate from a high IVGTT glucose/central obesity factor and a high triglyceride/low high density lipoprotein cholesterol factor. Together, these factors accounted for 55.9% of the total variance in the dataset. In conclusion, interindividual variations in plasma leptin concentrations are strongly related to the principal components of the insulin resistance syndrome. Further studies are needed to determine whether the insulin-leptin axis plays a coordinating role in this syndrome and whether plasma leptin concentrations could provide an additional measure of cardiovascular risk.
IL-1β–Induced Monocyte Chemoattractant Protein-1 Gene Expression in Endothelial Cells Is Blocked by Proteasome Inhibitors
Authors: Parry Graham C. N. , Martin Teresa , Felts Katherine A. , Cobb Ronald R.
Published: June 2011

Abstract—Human monocyte chemoattractant protein-1 (MCP-1) is expressed by a variety of cell types in response to various stimuli. MCP-1 expressed by the endothelium plays an important role in cell migration and activation. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we present evidence that the proteasome complex is involved in mediating the interleukin (IL)-1β induction of MCP-1 in endothelial cells. We present evidence that a proteasome inhibitor,N-acetyl-leucinyl-leucinyl-norleucinal (norLeu), and the protease inhibitor tosyl-Phe-chloromethylketone (TPCK) block IL-1β induction of MCP-1 protein expression. norLeu and TPCK also blocked IL-1β–induced MCP-1 promoter-driven reporter gene expression as well as nuclear factor (NF)-κB–mediated reporter gene expression. The effects of norLeu were due to its inhibition of the proteasome rather than calpain, because other calpain inhibitors had no effect on MCP-1 expression. In contrast to TPCK, which blocked NF-κB translocation to the nucleus, norLeu had no effect on NF-κB nuclear translocation or IL-1β–induced phosphorylation of p65. This study demonstrates that the proteasome pathway is involved in IL-1β–induced MCP-1 gene expression in human endothelial cells.