bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Distribution of 70-kD Heat Shock Protein in Atherosclerosis
Authors: Johnson A. Daniel , Berberian Paul A. , Tytell Michael , Bond M. Gene
Published: June 2011

AbstractSmooth muscle cell death may contribute to necrotic plaque rupture and subsequent thromboembolus. Stress-induced synthesis of heat-shock proteins (HSPs) normally protects cells from death, but vascular HSPs may become insufficient as cytotoxicity increases in advanced plaques. To determine whether vascular HSP content is altered near necrosis, we compared 70-kD HSP (HSP70) distribution between fibrotic and necrotic plaques in immunostained carotid endarterectomy specimens. Average levels of HSP70 immunoreactivity were compared by video densitometry between fibrotic and necrotic plaques or between their underlying media. Both necrotic plaques and their underlying media contained significantly more HSP70 staining than did fibrotic tissues. To test whether cellular HSP70 correlated with resistance to toxicity in vitro, aortic smooth muscle cells (aSMCs) were heat shocked to induce endogenous HSPs or given 2 to 50 μg/mL purified HSP70. Cells were then serum deprived or exposed to 12 to 96 μmol/L cholestanetriol (C3ol) or 25-hydroxycholesterol, and survival was determined. Cellular HSP70 content was assayed by immunoblotting, and protein synthesis was monitored by35S radiolabeling. Serum deprivation inhibited general protein synthesis but induced HSP70; C3ol exposure inhibited both overall protein and HSP70 synthesis, including post–heat shock. Induction of endogenous HSPs or 10 μg/mL exogenous HSP70 improved viability of serum-deprived cells (P<.05 andP<.01, respectively), while only exogenous HSP70 protected against C3ol (P<.002). The results suggest that insufficient HSP70 accumulates in aSMCs residing near necrosis to protect against plaque toxicity; aSMC death might then occur, allowing resident macrophages to degrade and destabilize the matrix, leading to rupture.
Loss of a Splice Donor Site at a ‘Skipped Exon’ in a Gene Homologous to Apolipoprotein(a) Leads to an mRNA Encoding a Protein Consisting of a Single Kringle Domain
Authors: Byrne Christopher D. , Schwartz Karen , Lawn Richard M.
Published: June 2011

Abstract
Apolipoprotein(a) [apo(a)] and plasminogen are located in a gene cluster on chromosome 6 together with two other genes that share highly homologous 5′ flanking regions. We have isolated the human liver transcript derived from one of these genes, designated apo(a)-related gene C, that encodes a polypeptide of 132 amino acids composed of a secretion signal and a single kringle domain. Although the gene encodes several additional kringle domains, sequence analysis shows that the second kringle is incomplete in the derived mRNA because it lacks an apparent exon present in the gene. Analysis of genomic sequence shows that the predicted exon at this site lacks a canonical splice donor site. This results in “exon skipping” during maturation of the mRNA, causing a coding frame shift and the presence of premature stop codons.
Magnetic Resonance Imaging–Assessed Adipose Tissue and Serum Lipid and Insulin Concentrations in Growth Hormone–Deficient Adults
Authors: Snel Yvonne E.M. , Doerga Manorath E. , Brummer Robert-Jan M. , Zelissen Pierre M.J. , Koppeschaar Hans P.F.
Published: June 2011

Abstract
The visceral and subcutaneous abdominal adipose tissue (AT) areas and the subcutaneous hip AT area were assessed by magnetic resonance imaging (MRI) in relation to serum lipid and plasma insulin levels in 12 growth hormone–deficient (GHD) adults before and after 6 months of replacement therapy with recombinant human growth hormone (rhGH) and in 12 healthy control subjects. Compared with control subjects, GHD patients had a significantly increased amount of visceral AT, which was inversely related with plasma HDL cholesterol and positively correlated with plasma triglyceride levels. Visceral AT was not associated with plasma total and LDL cholesterol or plasma insulin concentrations. GHD patients also had elevated serum total cholesterol, LDL cholesterol, and triglyceride levels compared with control subjects. After 6 months of rhGH replacement therapy the mean visceral, subcutaneous abdominal, and subcutaneous hip AT areas and serum concentration of total cholesterol decreased significantly, whereas serum HDL cholesterol concentration increased significantly. No significant correlations were found between changes in the amount of AT and changes in serum lipid and plasma insulin levels.
Increased Autoantibody Titers Against Epitopes of Oxidized LDL in LDL Receptor–Deficient Mice With Increased Atherosclerosis
Authors: Palinski Wulf , Tangirala Rajendra K. , Miller Elizabeth , Young Stephen G. , Witztum Joseph L.
Published: June 2011

Abstract
Increasing evidence indicates that immune processes modulate atherogenesis. Oxidized LDL (Ox-LDL) is immunogenic, and autoantibodies recognizing epitopes of Ox-LDL have been described in plasma and in atherosclerotic lesions of several species. To determine whether the titer of such autoantibodies correlates with the extent of atherosclerosis, we followed the development of antibodies against malondialdehyde-lysine, an epitope of Ox-LDL, in two groups of LDL receptor–deficient mice for 6 months. One group was fed an atherogenic diet (21% fat and 0.15% cholesterol) that resulted in marked hypercholesterolemia and extensive aortic atherosclerosis; the other group was fed regular rodent chow (4% fat) that did not alter plasma cholesterol levels and induced minimal atherosclerosis. Autoantibody titers significantly increased over time in the group on the atherogenic diet, whereas they remained constant in the chow-fed group. When data from both groups were pooled, a significant correlation was found between the autoantibody titers and the extent of atherosclerosis (
r
=.61,
P
<.01). Autoantibody titers also correlated with plasma cholesterol levels (
r
=.48,
P
<.05). These results suggest that the rise in autoantibody titers to an epitope of Ox-LDL in this murine model is partially determined by the extent of atherosclerosis but could also be influenced by the degree of hypercholesterolemia or other factors that may influence lipid peroxidation.
Transcriptional Activation of the Macrophage-Colony Stimulating Factor Gene by Minimally Modified LDL
Authors: Rajavashisth Tripathi B. , Yamada Hisashi , Mishra Nikhilesh K.
Published: June 2011

AbstractMinimally modified LDL (MM-LDL), obtained by mild iron oxidation or prolonged storage at 4°C, has been shown to induce the expression of macrophage-colony stimulating factor (M-CSF) in cultured aortic endothelial cells. To examine whether other cell types also respond to MM-LDL, we investigated its effect on the expression of M-CSF mRNA in mouse L-cells and human aortic smooth muscle cells. Both L-cells and human aortic smooth muscle cells showed increased levels of M-CSF mRNA in response to 10 to 200 μg/mL MM-LDL in a dose-dependent manner. This allowed us to use mouse L-cells as a model to study the mechanism involved in MM-LDL–mediated increase in M-CSF mRNA. Nuclear run-on assays showed that M-CSF gene transcription was activated by MM-LDL. In the present study, we identified specific elements that conferred MM-LDL–mediated transcriptional activation of the human M-CSF gene. Chimeric constructs containing sequential deletions in the 5′-promoter region of the M-CSF gene linked to a reporter chloramphenicol acetyltransferase (CAT) gene were transfected into mouse L-cells. The human M-CSF promoter region extending upstream from the transcription start site to nucleotide −406 showed maximum induction of CAT activity by MM-LDL. Induction of CAT activity was drastically reduced, with a deletion plasmid lacking the promoter region −406 to −344. A functional nuclear factor (NF)–κB binding site present in this critical region was required for MM-LDL–mediated induction of CAT activity since an internal deletion construct lacking this element showed significant loss of transcriptional activation. Similar results also were obtained with the use of bovine aortic endothelial cells, suggesting that part of the mechanism is shared in different cell types. Gel shift assays with bovine aortic endothelial cell nuclear extracts revealed that this element binds to MM-LDL–inducible nuclear protein(s) that exhibited DNA binding specificity of NF-κB and cross-reacted to NF-κB–specific antibodies. Taken together, these results are consistent with the involvement of NF-κB in the transcriptional activation of the human M-CSF gene by MM-LDL.
Glycosphingolipid Accumulation in the Aortic Wall Is Another Feature of Human Atherosclerosis
Authors: Mukhin Dmitry N. , Chao Fei-Fei , Kruth Howard S.
Published: June 2011

Abstract
High accumulation of lipids is a typical feature of an atherosclerotic lesion. We have previously identified the chemical structure of the major glycosphingolipids (GSLs) of human aorta; however, quantification of the absolute concentration of GSLs was not carried out. In the present study, for the first time we have performed a quantitative comparative analysis of GSL composition in the media and two sublayers of the intima taken from normal regions, fatty streaks, and atherosclerotic plaques of the human aorta. The intimal tissue containing fatty streaks and atherosclerotic plaques accumulated GSLs, predominantly glucosylceramide (GlcCer), lactosylceramide (LacCer), and ganglioside G
M3
. GSL levels in plaques were highest: GlcCer was 18- and 8-fold, LacCer was 8- and 7-fold, and G
M3
was 2.5- and 12-fold higher than in musculoelastic and elastic-hyperplastic intimal layers of normal regions, respectively. We did not observe a significant increase in other GSLs. An increase in the content of gangliosides G
D3
and G
D1a
was detected in the media underlying atherosclerotic lesions. On the basis of an analysis of the ratio of GlcCer, LacCer, and G
M3
accumulated in the tissue and cells of the elastic-hyperplastic layer of intima, we have concluded that the accumulation of the above-mentioned GSLs occurs mainly in the extracellular space of the intima. In this study, we have also demonstrated that extracellular lipid liposomes, which appear in the early stages of atherogenesis, are one locus of GSL accumulation in the extracellular space of the intima. The findings suggest that the GSL concentration and distribution within the normal and atherosclerotic aorta reflects a number of factors that include (1) synthesis of GSLs within the vessel wall, (2) deposition of GSLs within the vessel wall from plasma-derived lipoproteins, (3) the degree of association of the various GSLs with intimal cells as well as extracellular lipid particles, and (4) metabolic relationships between cholesterol and GSL accumulation.
Human Atherosclerotic Plaque Contains Both Oxidized Lipids and Relatively Large Amounts of α-Tocopherol and Ascorbate
Authors: Suarna Cacang , Dean Roger T. , May James , Stocker Roland
Published: June 2011

Abstract
We assessed the antioxidant status and contents of unoxidized and oxidized lipids in freshly obtained, homogenized samples of both normal human iliac arteries and carotid and femoral atherosclerotic plaque. Optimal sample preparation involved homogenization of human atherosclerotic plaque for 5 minutes, which resulted in recovery of most of the unoxidized and oxidized lipids without substantial destruction of endogenous vitamins C and E and 87% and 43% recoveries of added standards of α-tocotrienol and isoascorbate, respectively. The total protein, lipid, and antioxidant levels obtained from human plaque varied among donors, although the reproducibility of replicates from a single sample was within 3%, except for ubiquinone-10 and ascorbate, which varied by 20% and 25%, respectively. Plaque samples contained significantly more ascorbate and urate than control arteries, with no discernible difference in the vitamin C redox status between plaque and control materials. The concentrations of α-tocopherol and ubiquinone-10 were comparable in plaque samples and control arteries. However, approximately 9 mol percent of plaque α-tocopherol was present as α-tocopherylquinone, whereas this oxidation product of vitamin E was not detectable in control arteries. Coenzyme Q
10
in plaque and control arteries was only detected in the oxidized form ubiquinone-10, although coenzyme Q
10
oxidation may have occurred during processing. The most abundant of all studied lipids in plaque samples was free cholesterol, followed by cholesteryl oleate and cholesteryl linoleate (Ch18:2). Approximately 30% of plaque Ch18:2 was oxidized, with 17%, 12%, and 1% present as fatty acyl hydroxides, ketones, and hydroperoxides, respectively. In comparison, 7-ketocholesterol was detected at an ≈75-fold lower concentration. Normal arteries contained similar levels of protein as atherosclerotic arteries, much less free cholesterol, and no detectable amounts of unoxidized or oxidized cholesteryl esters. Together, these results demonstrate the coexistence in human plaque of large amounts of oxidized cholesteryl esters with significant concentrations of ascorbate and vitamin E in their reduced, antioxidant-active form. We conclude that compared with healthy human arteries, advanced atherosclerotic plaques are not deficient in the antioxidant vitamins C and E, despite the occurrence of massive lipid oxidation.
Inhibition of Hypercholesterolemia-Induced Atherosclerosis in the Nonhuman Primate by Probucol
Authors: Chang Mary Y. , Sasahara Masakiyo , Chait Alan , Raines Elaine W. , Ross Russell
Published: June 2011

Abstract
In nonhuman primates
(Macaca nemestrina)
treated with the antioxidant probucol during diet-induced hypercholesterolemia, intimal lesion area in the thoracic aorta was decreased, with increased resistance of plasma LDL to oxidation. The cellular and molecular changes associated with the decrease in lesion size in the probucol-treated hypercholesterolemic animals are quantitatively evaluated in this study. Lesions from the probucol-treated animals appear less mature and have altered lipid distribution. Abundant lipid-laden smooth muscle cells are found in the intima and media of the probucol-treated animals, with fewer medial lipid-laden macrophages, compared with lesions at similar sites in the control hypercholesterolemic animals. In both the control and probucol-treated animals, macrophages are the predominant cells in most lesions, but the ratio of macrophages to smooth muscle cells is decreased in the lower thoracic and upper abdominal aortic sites in the probucol-treated animals. Lesions at all aortic sites in the probucol-treated animals have a 35% to 80% reduction in the percentage of cells in cell cycle traverse, as indicated by immunostaining for proliferating cell nuclear antigen (% PCNA-positive). In both groups, macrophages and smooth muscle cells are PCNA-positive, but the majority (>60%) are macrophages. No difference in % PCNA-positive cells is seen in the iliac arteries, where the most advanced lesions were present at the time probucol administration was initiated. Limited Northern analysis of growth-regulatory molecules possibly involved in the cellular changes associated with lesions shows a 30% to 50% decrease in mRNA levels of platelet-derived growth factor (PDGF) B-chain, PDGF β-receptor, colony-stimulating factor type 1, and monocyte chemotactic protein 1. Thus, a potential role for an antioxidant such as probucol in the treatment of atherosclerosis may be to alter the early inflammatory fibroproliferative processes of the disease. Whether these effects are directly related to the antioxidant properties or some other activity of probucol is not yet known.
Relaxation of the Carotid Artery to Hypoxia Is Impaired in Watanabe Heritable Hyperlipidemic Rabbits
Authors: Taguchi Hisao , Faraci Frank M. , Kitazono Takanari , Heistad Donald D.
Published: June 2011

Abstract
We tested the hypothesis that relaxation of the carotid artery during hypoxia is mediated by activation of glibenclamide-sensitive potassium channels and that this response is impaired in hyperlipidemic rabbits. In New Zealand White rabbits (plasma cholesterol, 69±12 mg/dL, mean±SEM) and Watanabe heritable hyperlipidemic (WHHL) rabbits (plasma cholesterol, 677±99 mg/dL), tension of the carotid artery was measured in an organ bath under control conditions and during two levels of hypoxia. In normal rabbits, mild hypoxia produced 21±2% relaxation in arteries precontracted with phenylephrine. Removal of endothelium or the nitric oxide synthase inhibitor
N
G
-nitro-
l
-arginine (10
−4
mol/L) almost abolished relaxation in response to mild hypoxia in normal rabbits. Glibenclamide (10
−6
mol/L), an inhibitor of ATP-sensitive potassium channels, attenuated relaxation during mild hypoxia by almost 60%. In WHHL rabbits mild hypoxia relaxed the carotid artery by only 9±4% (
P
<.05 versus normal rabbits). Severe hypoxia produced greater relaxation of the carotid artery in normal than in WHHL rabbits (85±5% versus 52±8%, respectively,
P
<.05). Glibenclamide but not endothelial denudation or
N
G
-nitro-
l
-arginine attenuated relaxation during severe hypoxia in normal and WHHL rabbits. Relaxation of the carotid artery to sodium nitroprusside was similar in normal and WHHL rabbits. These findings suggest that relaxation of the carotid artery in response to mild and severe hypoxia is impaired in WHHL rabbits and is mediated, in large part, by activation of glibenclamide-sensitive potassium channels. Relaxation of the carotid artery in response to mild hypoxia is mediated primarily by endothelium-derived relaxing factor in normal rabbits and impairment of response to mild hypoxia in WHHL is probably secondary to endothelial dysfunction.
Increased Mitogenic Response to Heparin-Binding Epidermal Growth Factor–like Growth Factor in Vascular Smooth Muscle Cells of Diabetic Rats
Authors: Fukuda Kazuto , Inui Yoshiaki , Kawata Sumio , Higashiyama Shigeki , Matsuda Yukihiko , Maeda Yuichi , Igura Takumi , Yoshida Shingo , Taniguchi Naoyuki , Matsuzawa Yuji
Published: June 2011

Abstract
We investigated the mitogenic effects of heparin-binding epidermal growth factor–like growth factor (HB-EGF) in vascular smooth muscle cells (SMCs) obtained from rats with streptozotocin (STZ)-induced diabetes and evaluated the role of heparan sulfate proteoglycan (HSPG) in inducing these effects. HB-EGF significantly increased DNA synthesis in the SMCs of diabetic rats (STZ-SMCs) compared with control rats (control SMCs). However, the mitogenic effects of EGF, which shares EGF receptors with HB-EGF, and basic fibroblast growth factor, another heparin-binding growth factor, were similar in STZ-SMCs and control SMCs. The mitogenic response to HB-EGF in SMCs of insulin-treated diabetic rats was similar to the response in control SMCs. HB-EGF–induced autophosphorylation of EGF receptors was increased in STZ-SMCs compared with control SMCs, although the number of EGF receptors in STZ-SMCs was 40% of that in controls. This increased mitogenic response to HB-EGF in STZ-SMCs was completely inhibited by treatment with heparitinase, chlorate, and a synthetic peptide corresponding to the heparin-binding domain of HB-EGF. Compared with heparan sulfate isolated from control SMCs, heparan sulfate isolated from STZ-SMCs was of smaller molecular size and caused a greater mitogenic effect of HB-EGF. These findings suggest that the mitogenic response to HB-EGF is increased in SMCs of diabetic rats. Changes in cell-associated heparan sulfate in STZ-SMCs may be related to the increased mitogenic response to HB-EGF.
Inhibitors for the In Vitro Assembly of Lp(a)
Authors: Frank Sasa , Durovic Srdan , Kostner Karam , Kostner Gert M.
Published: June 2011

Abstract
Lp(a) is composed of an LDL-like core and the glycoprotein apo(a). Current evidence strongly suggests that the assembly of this atherogenic lipoprotein proceeds outside the liver cells in a two-step fashion. In the first step, a loose complex is formed involving kringle-4 motifs in apo(a) and one or more Lys side chains in apoB-100. In the second step, this complex is stabilized by a disulfide bridge. Indications are that Lp(a) assembly is critical in the determination of plasma apo(a) concentrations. Therefore, we searched for substances that interfere with the first step of Lp(a) assembly. ε-Aminohexoic acid (ε-AHA), known as an inhibitor from earlier assembly studies, had an IC
50
of 4.8 mmol/L. The IC
50
of Pro, HO-p-aminobenzene sulfonamide, Lys,
N
-ε-acetyl-Lys, taurine, Glu, serotonin, and benzamidine were all >20 mmol/L. γ-Aminobutyric acid, spermine, and spermidine exhibited IC
50
on the same order of magnitude as ε-AHA. The substances with the highest inhibitory action were tranexamic acid and δ-aminovaleric acid. Seven of eight patients treated in a pilot study with tranexamic acid (Cyclocapron) responded with a decrease of plasma apo(a) of 18.5±8.2%. We suggest that substances that interfere with the Lp(a) assembly are worth pursuing further for their usefulness as therapeutic agents in reducing high plasma Lp(a) concentrations.
Insulin Sensitivity, Lipids, and Blood Pressure in Young American Blacks
Authors: Falkner Bonita , Kushner Harvey , Tulenko Thomas , Sumner Anne E. , Marsh Julian B.
Published: June 2011

Abstract
The purpose of this study was to determine whether insulin resistance was linked with alterations in plasma lipids in adult young blacks with borderline hypertension. Ninety-four American blacks participated (46 men, 48 women, age range 28 to 33 years). Within this group of 94 subjects, there were 60 normotensive (Nt) subjects and 36 subjects with borderline hypertension (BHt), defined as blood pressure >135/85 mm Hg. None of the subjects were diabetic or receiving antihypertension medication. All participants had blood pressure and anthropometric measurements, a fasting lipid profile, an oral glucose tolerance test, and a euglycemic hyperinsulinemic clamp. Insulin-stimulated glucose utilization (M), determined by insulin clamp, was significantly lower in the BHt subjects compared with the Nt subjects (men, Nt 6.91±0.62 versus BHt 5.54±0.65; women, Nt 5.97±0.47 versus BHt 3.79±0.38 mg · kg
−1
· min
−1
,
P
=.006). When M was corrected for adiposity and expressed in milligrams per kilogram of fat free mass (M′), the difference between Nt and BHt remained significant (
P
=.006). There was a significant correlation of M′ with systolic blood pressure (
r
=−.393,
P
<.0001), HDL-C (
r
=.382,
P
<.0001), triglyceride level (
r
=−.308,
P
<.001), apolipoprotein A-I (
r
=.190,
P
=.033), and apolipoprotein B (
r
=−.277,
P
=.004). When all lipid variables were entered in a stepwise multiple linear regression analysis, HDL-C emerged as the most significant lipid component in the model for insulin resistance. These data suggest that in American blacks with mild hypertension, the risk for cardiovascular disease may be augmented in the presence of insulin resistance.
Serum Paraoxonase Activity, Concentration, and Phenotype Distribution in Diabetes Mellitus and Its Relationship to Serum Lipids and Lipoproteins
Authors: Abbott Caroline A. , Mackness Michael I. , Kumar Sudhesh , Boulton Andrew J. , Durrington Paul N.
Published: June 2011

AbstractHuman serum paraoxonase is physically associated with HDL and has been implicated in the detoxification of organophosphates and possibly in the prevention of LDL lipid peroxidation. We investigated the serum activity and concentration of paraoxonase in 78 patients with type 1 diabetes mellitus, 92 with type 2 diabetes, and 82 nondiabetic control subjects. Paraoxonase activity was generally lower in diabetics than in control subjects. This decrease was unrelated to differences in paraoxonase phenotype distribution or its serum concentration. Rather, the difference in paraoxonase activity was explained by its specific activity, which was lower in diabetics, indicating either the presence of a circulating inhibitor or disturbance of the interaction of paraoxonase with HDL affecting its activity. Paraoxonase specific activity was lowest in patients with peripheral neuropathy, suggesting an association of paraoxonase with neuropathy. In control subjects but not patients with diabetes, paraoxonase correlated with HDL cholesterol and apolipoprotein A-1. Our results indicate that the low paraoxonase activity in diabetes is due to decreased specific activity. In other studies low serum paraoxonase activity has been associated with increased susceptibility to atherosclerosis, and the present results also suggest an association with peripheral neuropathy, which could be due to reduced capacity to detoxify lipid peroxides in diabetes.
Intravenous Injection of Rabbit Apolipoprotein A-I Inhibits the Progression of Atherosclerosis in Cholesterol-Fed Rabbits
Authors: Miyazaki Akira , Sakuma Shin , Morikawa Wataru , Takiue Takanori , Miake Fumio , Terano Tsuyoshi , Sakai Masakazu , Hakamata Hideki , Sakamoto Yu-Ichiro , Naito Makoto , Ruan Yingmao , Takahashi Kiyoshi , Ohta Takao , Horiuchi Seikoh
Published: June 2011

Abstract
The effects of intravenous injection of purified rabbit apoA-I on the progression of aortic atherosclerosis in cholesterol-fed rabbits were examined. In experiment 1, 28 rabbits were equally divided into groups A and B and fed a 0.5% cholesterol diet for 90 days. For the last 30 days, group B received 40 mg apoA-I every week. The fatty streak lesions in group B (23.9±15.6%) were significantly suppressed compared with those in group A (46.0±24.9%) (
P
<.05). In experiment 2, 33 rabbits were divided into four groups (8 or 9 rabbits per group) and fed a 0.5% cholesterol diet. Group A was killed on day 105, while groups B, C, and D were maintained for an additional 60 days on a normal diet, during which time groups C and D received 1 mg apoA-I every other day or 40 mg apoA-I every week, respectively. The lesions in group C (70.2±15.4%) and group D (65.7±20.0%) were significantly suppressed compared with those in group B (86.2±13.7%) (
P
<.05) but were not reduced to the level of group A (50.0±22.9%). Although apparent regression was not observed under these conditions, the present study provided the first evidence for the antiatherogenic effect of homologous apoA-I on the progression of atherosclerosis in cholesterol-fed rabbits.
Overexpression of Human Apolipoprotein B-100 in Transgenic Rabbits Results in Increased Levels of LDL and Decreased Levels of HDL
Authors: Fan Jianglin , McCormick Sally P.A. , Krauss Ronald M. , Taylor Stacy , Quan Ricky , Taylor John M. , Young Stephen G.
Published: June 2011

Abstract
In this study, an 80-kb human genomic DNA fragment spanning the human apoB gene was used to generate transgenic New Zealand White rabbits that expressed human apoB-100. The concentration of human apoB in the plasma of the transgenic rabbits ranged between 5 and 100 mg/dL. The transgenic rabbits had nearly threefold elevations in the plasma levels of triglycerides and cholesterol compared with nontransgenic controls. Nearly all the cholesterol and human apoB in the plasma was in the LDL fraction. Pronounced triglyceride enrichment of the LDL fraction was a striking feature of human apoB overexpression in the transgenic rabbits, in which the LDL fraction contained more than 75% of the plasma triglycerides. The triglyceride-enriched LDL particles were smaller and more dense than the native rabbit LDL and contained markedly increased amounts of apoE and apoC-III. In the nontransgenic control animals most of the triglycerides were in the VLDL, and most of the apoE and apoC-III were in the VLDL and HDL fractions. In addition to increased LDL levels, overexpression of human apoB in rabbits resulted in lower plasma levels of HDL cholesterol and apoA-I. In our prior studies on transgenic mice expressing human apoB, we documented triglyceride-rich LDL and reduced levels of HDL cholesterol. These prior findings in mice, together with the present findings in transgenic rabbits, suggest that triglyceride-rich LDL and lowered levels of HDL cholesterol may be hallmark features of apoB overexpression.
Mechanism of Inhibition of Neointimal Formation by the Angiotensin-Converting Enzyme Inhibitor Cilazapril
Authors: Fingerle Jürgen , Müller Rita M.K. , Kuhn Herbert , Pech Michael , Baumgartner Hans Rudolf
Published: June 2011

Abstract
We investigated the mechanism of inhibition of neointima formation by the angiotensin-converting enzyme inhibitor cilazapril in a rat model of balloon-catheter injury in the carotid artery. We looked for the effects of cilazapril on all phases of the response to injury, ie, on proliferation of smooth muscle cells (SMCs) in the media, their migration, their proliferation in the neointima, and their deposition of extracellular matrix in the neointima. Although treatment was discontinued after 2 weeks, the inhibitory effect of cilazapril on neointimal formation was evident even 52 weeks after injury. The amount of extracellular matrix deposited in the intima during cilazapril treatment was decreased by 20% 2 weeks after injury, but no effect was seen if tissues were analyzed at 4 or 52 weeks. [
3
H]Thymidine-labeled cells (pulse labeling as well as 14-day continuous labeling) showed a decrease in SMC labeling in the tunica media by 50%, but no inhibition in the labeling indices was seen in the neointima. The fraction of unlabeled neointimal cells in the cilazapril-treated rats as judged from continuous labeling experiments was inhibited by 86%. Taken together, these data suggest an antiproliferative effect on medial SMCs and an inhibition of SMC migration into the intima by cilazapril. Since intimal extracellular matrix deposition was only delayed, the decrease in medial SMC proliferation and subsequent migration seems to be the main reason for the reduction of neointima formation.
Lipoproteins Regulate C-Type Natriuretic Peptide Secretion From Cultured Vascular Endothelial Cells
Authors: Sugiyama Seigo , Kugiyama Kiyotaka , Matsumura Toshiyuki , Suga Shin-ichi , Itoh Hiroshi , Nakao Kazuwa , Yasue Hirofumi
Published: June 2011

Abstract
We have shown that oxidized low-density lipoprotein (Ox-LDL) modulates various endothelial cell (EC) functions. C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family to be discovered, is secreted from peripheral vascular ECs and regulates body fluid homeostasis, vascular tone, and vascular growth. This study was designed to investigate the effects of lipoproteins on CNP secretion from cultured ECs. Treatment of bovine carotid ECs with Ox-LDL and its extracted lipids resulted in a concentration-dependent suppression of the spontaneous and transforming growth factor-β1–stimulated secretion of CNP. Native LDL, its extracted lipids, and acetylated LDL were inactive. Ox-LDL depleted of its amphiphilic lipids, which was prepared by incubation with defatted albumin, lost its suppressive effect on CNP secretion. 7-Ketocholesterol, one of the amphiphilic lipids in Ox-LDL that is transferable from Ox-LDL to defatted albumin, suppressed CNP secretion by ECs, thus mimicking the effect of Ox-LDL. Coincubation with high-density lipoprotein (HDL), which alone had no effect on CNP release, significantly prevented Ox-LDL–induced inhibition of CNP secretion by ECs. Analysis by thin-layer chromatography demonstrated that oxysterols, including 7-ketocholesterol, in Ox-LDL were transferred from Ox-LDL to HDL during coincubation of these two lipoproteins. These results indicate that Ox-LDL suppresses CNP secretion from ECs by 7-ketocholesterol or other transferable hydrophilic lipids in Ox-LDL, and the suppressive effect of Ox-LDL is reversed by HDL. Lipoproteins thus may regulate CNP secretion from the endothelium of atherosclerotic arteries.
High-Density Lipoproteins Inhibit Cytokine-Induced Expression of Endothelial Cell Adhesion Molecules
Authors: Cockerill Gillian W. , Rye Kerry-Anne , Gamble Jennifer R. , Vadas Mathew A. , Barter Philip J.
Published: June 2011

AbstractWhile an elevated plasma concentration of HDLs is protective against the development of atherosclerosis and ensuing coronary heart disease (CHD), the mechanism of this protection is unknown. One early cellular event in atherogenesis is the adhesion of mononuclear leukocytes to the endothelium. This event is mediated principally by vascular cell adhesion molecule-1 (VCAM-1) but also involves other molecules, such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin. We have investigated the effect of isolated plasma HDLs and reconstituted HDLs on the expression of these molecules by endothelial cells. We show that physiological concentrations of HDLs inhibit tumor necrosis factor-α (TNF-α) or interleukin-1 (IL-1) induction of these leukocyte adhesion molecules in a concentration-dependent manner. Steady state mRNA levels of TNF-α–induced VCAM-1 and E-selectin are significantly reduced by physiological concentrations of HDLs. At an HDL concentration of 1 mg/mL apolipoprotein A-I, the protein expressions of VCAM-1, ICAM-1, and E-selectin were inhibited by 89.6±0.4% (mean±SD, n=4), 64.8±1.0%, and 79.2±0.4%, respectively. In contrast, HDLs have no effect on the expression of platelet endothelial cell adhesion molecule (PECAM) or on the expression of the p55 and p75 subunits of the TNF-α receptor. HDLs were effective when added from 16 hours before to 5 minutes after cytokine stimulation. HDLs had no effect on TNF-α–induced expression of ICAM-1 by human foreskin fibroblasts, suggesting that the effect is cell-type restricted. This study provides the first evidence that HDLs may protect against CHD by inhibiting the expression of adhesion molecules, which are required for the interaction between leukocytes and the endothelium.
Expression of the Macrophage Scavenger Receptor in Atheroma
Authors: Geng Yong-jian , Holm Jan , Nygren Stina , Bruzelius Martin , Stemme Sten , Hansson Göran K.
Published: June 2011

AbstractScavenger receptors mediate internalization of modified lipoproteins and foam cell transformation of monocyte-derived macrophages. Their expression is independent of the intracellular cholesterol content but is modulated by immune-derived cytokines. We investigated macrophage scavenger receptor (MSR) expression in monocyte-macrophages from human peripheral blood and in atherosclerotic lesions and analyzed its relationship to T lymphocytes and immunoregulatory cytokines by immunohistochemistry and polymerase chain reaction (PCR). Antibodies specific for the two MSR isoforms were generated by immunizing rabbits with isoform-specific synthetic peptides conjugated to keyhole limpet hemocyanin. In human atherosclerotic plaques, these antibodies stained macrophages and foam cells in a pattern that corresponded to the distribution of the macrophage marker CD68. CD3-positive T cells and α-actin–positive smooth muscle cells exhibited no reactivity to the anti-MSR antibodies. The frequency of cells stained with antibodies to MSR type I was equal to that of cells stained for type II, suggesting that most macrophages coexpress both isoforms. Reverse transcription (RT)–PCR analysis confirmed that both MSR isoforms were expressed in all plaques examined. There was, however, a tendency toward a lower immunohistochemical staining intensity for MSR type I and a decreased number of lipid-rich foam cells in T cell–rich areas. The mRNAs for interleukin-2 and interferon-γ, two major products of activated T cells, were detected by RT-PCR in all plaques tested. This indicates that activation of T lymphocytes occurs in atherosclerotic plaques. Since interferon-γ downregulates MSR expression, these observations suggest a potential mechanism for local regulation of MSR expression in the atherosclerotic plaque.
Oxidized Low-Density Lipoproteins Facilitate Leukocyte Adhesion to Aortic Intima Without Affecting Endothelium-Dependent Relaxation
Authors: Mehta Asha , Yang Baichun , Khan Saeed , Hendricks James B. , Stephen Claudia , Mehta Jawahar L.
Published: June 2011

Abstract
Inflammatory cell deposition in atherosclerotic blood vessels has been thought to relate to loss of endothelium-derived nitric oxide (NO). To examine whether cell deposition correlates temporally with the loss of NO activity, rat aortic rings were incubated with buffer, native LDL (n-LDL), oxidized LDL (ox-LDL), or the endothelium-derived relaxing factor synthase inhibitor
N
ω
-nitro-
l
-arginine methyl ester (
l-
NAME) for 2 hours, and vascular contractile response to norepinephrine and relaxant response to acetylcholine, thrombin, and calcium ionophore A23,187 were examined. Thereafter, the rings were exposed to biotin-fluorescein isothiocyanate-labeled fluorescent or unlabeled leukocytes for 30 minutes. Cell adhesion was quantitated by fluorescent microscopy as well as by scanning electron microscopy. Incubation with n-LDL or ox-LDL did not affect either the contractile or the relaxant response of rings. However, leukocyte adhesion increased markedly in all ox-LDL–treated rings but not in those treated with n-LDL. Thus, leukocyte adhesion occurred independent of NO activity. In keeping with this concept, pretreatment of rings with the NO precursor
l
-arginine failed to influence leukocyte adhesion to rings incubated with ox-LDL. Treatment of rings with
l-
NAME also resulted in adhesion of a large number of leukocytes. Furthermore, all rings treated with ox-LDL or
l-
NAME demonstrated marked expression of P-selectin leukocyte adhesion molecules, determined by immunohistochemistry. Pretreatment of rings with the P-selectin blocking antibody PB1.3 markedly decreased deposition of leukocytes in rings exposed to ox-LDL. These data show that cell adhesion to vascular intima exposed to ox-LDL shows no temporal relation with attenuation of NO activity, although inhibition of NO synthesis leads to leukocyte deposition. P-selectin expression on vascular rings exposed to ox-LDL appears to be the basis of leukocyte deposition.
Abnormal Reverse Cholesterol Transport in Controlled Type II Diabetic Patients
Authors: Cavallero Elisabeth , Brites Fernando , Delfly Bernard , Nicolaı̈ew Nathalie , Decossin Christelle , De Geitere Catherine , Fruchart Jean-Charles , Wikinski Regina , Jacotot Bernard , Castro Graciela
Published: June 2011

Abstract
The high incidence and prevalence of coronary heart disease in diabetes mellitus is clearly established. The usual lipid pattern found in type II diabetic patients is a moderate increase in fasting triglyceride levels associated with low HDL cholesterol levels. These abnormalities are further amplified in the postprandial state. To study the effect of these alterations on reverse cholesterol transport, we isolated lipoprotein containing apoA-I but not apoA-II (LpA-I) particles by immunoaffinity chromatography from the plasma of well-controlled type II diabetic patients and nondiabetic matched control subjects. Different parameters involved in this antiatherogenic pathway were measured in both fasting and postprandial states. Diabetic patients had reduced levels of LpA-I particles that were protein enriched and phospholipid depleted. Gradient gel electrophoresis showed that control LpA-I particles had five distinct populations, whereas diabetic particles lacked the largest one. LpA-I isolated from diabetic plasma exhibited a decreased capacity to induce cholesterol efflux from Ob 1771 adipose cells both in fasting (15.1±10.0% versus 7.5±2.7%,
P
<.05) and postprandial (17.7±11.2% versus 7.7±3.9%,
P
<.05) states, whereas only control particles showed significantly higher ability to promote cholesterol efflux after the test meal (
P
=.02). Lecithin:cholesterol acyltransferase activity measured with an exogenous substrate showed a 54% increase and an 18% decrease postprandially for control subjects and patients, respectively. Thus, the different abnormalities found in the fasting state were further amplified in the postprandial situation. This resulted in LpA-I particles with aberrant size and composition and decreased ability to accomplish their antiatherogenic role in type II diabetic patients.
Ten LDL Receptor Mutants Explain One Third of Familial Hypercholesterolemia in a German Sample
Authors: Schuster Herbert , Keller Christiane , Wolfram Günther , Zöllner Nepomuk
Published: June 2011

Abstract
Mutational defects in the LDL receptor are responsible for familial hypercholesterolemia (FH); thus far more than 150 mutations have been described. Nevertheless, systematic searches among the Germans have not been conducted. We used single-strand conformational polymorphism and polymerase chain reaction to find mutations in 10 index patients and in 40 other individuals with heterozygous FH. Our screen in the 10 index patients revealed 7 hitherto undescribed mutations. A screen of the 40 additional FH patients disclosed 20 defective of 54 total alleles and allowed specific diagnoses in 88 family members. We also found two families in which the children were markedly affected by FH, but the expected parental expression of the trait was not manifest. This observation suggests a role for additional environmental and genetic influences. Our report represents the first comprehensive effort to identify FH mutations in Germany. We found 10 mutations and these mutations explain 37% of FH cases. Our data may have relevance to expected FH patterns in central Europe.
Effect of Selective Factor Xa Inhibition on Arterial Thrombus Formation Triggered by Tissue Factor/Factor VIIa or Collagen in an Ex Vivo Model of Shear-Dependent Human Thrombogenesis
Authors: Ørvim Una , Barstad R. Marius , Vlasuk George P. , Sakariassen Kjell S.
Published: June 2011

Abstract
Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of factor Xa. TAP has shown good antithrombotic efficacy in experimental animal models of disseminated intravascular coagulation and venous and arterial thrombogenesis. In the present study we evaluated the effect of recombinant TAP (rTAP) on acute thrombus formation in human nonanticoagulated blood triggered either by tissue factor (TF) or by collagen at arterial shear conditions. The main goal was to establish the role of factor Xa in thrombus formation by use of an optimal inhibitory concentration of rTAP. Blood was drawn directly from an antecubital vein by a pump over the respective thrombogenic surfaces, which were positioned in a parallel-plate perfusion chamber. rTAP was mixed homogeneously into the flowing blood by a heparin-coated device positioned proximal to the perfusion chamber. The passage of blood through this device caused minor activation of coagulation but little activation of platelets. Fibrinopeptide A and β-thromboglobulin levels after 5 minutes of blood perfusion were, on average, 14 ng/mL and 45 IU/mL, respectively. rTAP at a plasma concentration of 0.90 μmol/L completely inhibited TF/factor VIIa–dependent thrombus formation at wall shear rates of 650 and 2600 s
−1
. These shear conditions are comparable to those in medium-sized arteries and in moderately stenosed small arteries, respectively. In contrast to the TF-coated surface, rTAP was less efficient in reducing collagen-induced thrombus formation. While a significant reduction of 53% was observed at 650 s
−1
, thrombus formation at 2600 s
−1
was not affected by rTAP. Thus, rTAP is an efficient inhibitor of thrombin-driven human thrombus formation on the TF-rich surface but less efficient when thrombus formation is elicited by type III collagen. The lack of antithrombotic effect on collagen type III at 2600 s
−1
corroborates earlier findings, showing that collagen-induced thrombus formation in blood from patients with severe factor VIII deficiency is not affected at this blood flow condition and thus is not dependent on the prothrombotic effects of thrombin.
Increased Fibrinogen Levels in the Offspring of Hypertensive Men
Authors: Válek Jiří , Válková Libuše , Vlasáková Zuzana , Topinka Vladimír
Published: June 2011

Abstract
Epidemiological studies have revealed that elevated fibrinogen concentrations are associated with an increased risk of myocardial infarction, stroke, intermittent claudication, and cardiovascular mortality. The manner in which fibrinogen operates in atherogenesis has not yet been elucidated, but genetic control of fibrinogen levels is partially responsible. Fibrinogen frequently acts in concert with hyperlipidemia, diabetes, hypertension, physical inactivity, and age, variables that are influenced by insulin action. Because the offspring of hypertensive men tend to be hyperinsulinemic and insulin resistant from a young age, we hypothesized that their increased fibrinogen levels might reflect decreased insulin action and thus play a role in the metabolic syndrome. We chose 48 adult offspring (mean age, 38.4 years) of 30 fathers who had been treated for hypertension, and the former were matched by age, body mass index, sex, and smoking habits with 37 control subjects. Elevations in fibrinogen concentration (3.63±0.93 versus 2.87±0.54 g/L,
P
<.001) paralleled increases in blood glucose and insulin levels, estimates of insulin resistance, and blood pressure. In the offspring, in contrast to the control group, correlations between fibrinogen and metabolic-syndrome variables (ie, insulin, glucose, and waist and hip circumferences) were found. In stepwise multiple regression analyses, age and smoking habits were entered as variables in both study groups, but postload insulin and high-density lipoprotein cholesterol were entered as variables in the offspring group only. We propose that familial predisposition influences the relationship between insulin concentration and fibrinogen, an effect that may contribute to the clinical importance of the metabolic syndrome.
Quantitative Analysis of Repeat Adenovirus-Mediated Gene Transfer Into Injured Canine Femoral Arteries
Authors: Ueno Hikaru , Li Jian-Jun , Tomita Hideharu , Yamamoto Hiroaki , Pan Yan , Kanegae Yumi , Saito Izumu , Takeshita Akira
Published: June 2011

Abstract
We quantitatively evaluated the effectiveness of a repeat administration of a recombinant adenoviral vector expressing bacterial
Escherichia coli lacZ
into the same arterial site of a relatively large animal, the dog. The replication-defective adenoviral vector was introduced percutaneously into balloon-injured femoral arteries through a double-balloon catheter. After a single dose of adenoviral vector, up to 90% of surface (73±16%, n=7) and smooth muscle cells in multiple layers of the media showed transgene expression as evaluated by 5-bromo-4-chloro-3-indoyl β-
d
-galactopyranoside histostaining without extralocal expression, as assessed by polymerase chain reaction. High-level expression (measured as β-galactosidase activity) peaked 7 days after transfer and was transient, although it was retained for a month. Second doses of the same adenovirus to the same arterial site were given 1, 2, 5, or 8 weeks after the first administration. At 1 week the second dose significantly enhanced
lacZ
expression. At 2, 5, or 8 weeks the second dose reinduced
lacZ
expression at 25% to 30% of the full expression.
lacZ
expression was also detected in preimmuned dogs, although the expression levels correlated inversely to the titer of neutralizing antibodies in their serum. These results demonstrate that arterial gene expression can be enhanced by a second administration of the same adenovirus after a short interval and that a repeat dose after a long interval partially but significantly reinduces gene expression despite the presence of an immune response. These data may provide an additional scientific foundation for the use of adenovirus-mediated arterial gene transfer in future clinical practice.