bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced collagen-induced responses of platelets from rabbits with diet-induced hypercholesterolemia are due to increased sensitivity to TxA2. Response inhibition by chronic ethanol administration in hypercholesterolemia is due to reduced TxA2 formation.
Authors: Latta E K , Packham M A , Gross P L , Rand M L
Published: June 2011

The effects of dietary cholesterol and chronic administration of moderate amounts of ethanol on collagen-induced platelet responses were investigated. Three groups of rabbits were fed the following diets for 8 weeks: a normal chow diet, a cholesterol-enriched (0.25% wt/wt) chow diet, and a cholesterol-enriched chow diet plus 6% ethanol in the drinking water for the final week of the dietary period. Cholesterol feeding enhanced collagen-induced responses-aggregation, secretion of [14C]serotonin from prelabeled platelets, and thromboxane formation--of suspensions of washed platelets, and chronic ethanol treatment significantly reduced these enhanced responses. These effects are mediated by thromboxane A2 (TxA2) rather than ADP. Experiments with collagen-stimulated platelets in which feedback amplification of TxA2 was blocked with the prostaglandin H2/TxA2 receptor blocker BM 13.177 and experiments with aspirin-treated platelets stimulated with the stable TxA2 mimetic U46619 showed that cholesterol feeding enhanced platelet sensitivity to TxA2 rather than formation of TxA2 by platelets that had interacted with collagen. Without BM 13.177 or aspirin, TxA2 increased the amount of TxA2 formed by feedback amplification. In contrast, decreased responsiveness to collagen by platelets from cholesterol-fed rabbits given ethanol was due to inhibition of TxA2 formation rather than reduced sensitivity to TxA2. Platelets from cholesterol-fed rabbits given ethanol did not develop tolerance to the acute inhibitory effects of ethanol. Our results indicate that administration of moderate amounts of ethanol to cholesterol-fed rabbits inhibits enhanced collagen-induced responses of platelets by a TxA2-dependent pathway that involves reduction of TxA2 formation rather than reduction of platelet responses to TxA2.
Platelet calcium homeostasis is abnormal in patients with severe arteriosclerosis.
Authors: Vicari A M , Monzani M L , Pellegatta F , Ronchi P , Galli L , Folli F
Published: June 2011

To evaluate platelet calcium homeostasis in a typical thrombosis-prone clinical condition, 14 patients with severe arteriosclerosis and 11 healthy control subjects were studied. Platelet intracellular free calcium concentration ([Ca2+]i) was evaluated by means of the fluorescent probe fura 2 under resting conditions and after challenge with 0.05, 0.1, and 0.5 U/mL thrombin (final concentrations). Three different concentrations of extracellular ionized calcium ([Ca2+]e) were used: 1 mmol/L, 1 mumol/L, and < 1 nmol/L. Resting platelet [Ca2+]i was significantly higher (P < .001) in patients than in control subjects. After addition of 0.05 U/mL thrombin, the relative increase of [Ca2+]i was lower in patients than in control subjects in each of the three [Ca2+]e conditions (P = .05 at 1 mmol/L, P = .02 at 1 mumol/L, and P = .04 at < 1 nmol/L). After addition of 0.1 U/mL thrombin, the relative increase of [Ca2+]i was lower in patients than in control subjects under two [Ca2+]e conditions, 1 mumol/L and < 1 nmol/L (P = .04 and P = .03 respectively). With 0.5 U/mL thrombin, a trend toward lower values in patients than in control subjects was observed, reaching statistical significance (P = .03) only at < 1 nmol/L [Ca2+]e. These results suggest that calcium homeostasis is abnormal in platelets from patients with severe arteriosclerosis and probably reflects a chronic activation.
Fish oil improves arterial compliance in non-insulin-dependent diabetes mellitus.
Authors: McVeigh G E , Brennan G M , Cohn J N , Finkelstein S M , Hayes R J , Johnston G D
Published: June 2011

In a double-blind, placebo-controlled study we investigated the effects of dietary fish oil supplementation on arterial wall characteristics in 20 patients with non-insulin-dependent diabetes mellitus. Estimates reflecting compliance values in the large arteries and more peripheral vasculature, as measured by pulse-contour analysis, improved significantly after 6 weeks of fish oil therapy compared with values recorded at baseline and after 6 weeks' administration of olive oil. The large-artery compliance estimate increased from 1.50 (confidence interval [CI], 1.31 to 1.69) mL/mm Hg at baseline to 1.68 (CI, 1.52 to 1.84) mL/mm Hg after fish oil administration (P < .01). The oscillatory compliance value increased from 0.015 (CI, 0.011 to 0.019) mL/mm Hg at baseline to 0.022 (CI, 0.016 to 0.028) mL/mm Hg after fish oil ingestion (P < .05). No changes occurred in arterial blood pressure, cardiac output, stroke volume, or systemic vascular resistance with either intervention. The improved compliance estimates with fish oil ingestion occurred without altering fasting blood glucose and cholesterol concentrations. These results support the hypothesis that fish oils alter vascular reactivity and favorably influence arterial wall characteristics in patients with non-insulin-dependent diabetes mellitus. These direct vascular effects, expressed at the level of the vessel wall, may contribute to the cardioprotective actions of fish oil in humans.
Expression of LDL receptor-related protein/alpha 2-macroglobulin receptor in human normal and atherosclerotic arteries.
Authors: Lupu F , Heim D , Bachmann F , Kruithof E K
Published: June 2011

Low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR) is a multifunctional cell-surface receptor that is responsible for the clearance of lipoprotein remnants, proteases, or cytokines/growth factors in complex with alpha 2-macroglobulin as well as of plasminogen activators complexed with inhibitors. We investigated the expression of LRP/alpha 2-MR in healthy and atherosclerotic human arteries by in situ hybridization using an LRP/alpha 2-MR mRNA-specific riboprobe and immunocytochemistry using specific monoclonal antibodies. The cell types expressing LRP/alpha 2-MR were identified by immunolabeling of antigens specific for endothelial cells, smooth muscle cells, and macrophages. In normal arteries, LRP/alpha 2-MR mRNA and protein were found in smooth muscle cells of the media and vasa vasorum and in adventitial fibroblasts. Endothelial cells were negative for LRP/alpha 2-MR protein but positive for its mRNA. Atherosclerotic arteries exhibited a strong labeling for LRP/alpha 2-MR mRNA and protein that was observed in intimal smooth muscle cells exhibiting normal or foam cell characteristics and in lipid-laden cells positive for macrophage markers. A particularly high expression was detected in macrophages located in the cap of the lipid-rich necrotic core. These results suggest that cellular components of the atherosclerotic plaque express LRP/alpha 2-MR. This receptor may play an important local scavenger role for lipoprotein remnants, for growth factors/cytokines, and for extracellular protease-inhibitor complexes.
Evidence Suggestive of Activation of the Intrinsic Pathway of Blood Coagulation After Injection of Factor Xa/Phospholipid Into Rabbits
Authors: Warn-Cramer Bonnie J. , Rapaport Samuel I.
Published: June 2011

Abstract
The present study was carried out to extend an earlier observation from this laboratory that mean plasma factor X levels fell by about 15% after the injection into rabbits of a formed factor Xa/phospholipid complex that caused only minimal intravascular coagulation. We have now injected rabbits with formulations of factor Xa/phospholipid that caused considerable intravascular coagulation, as documented by substantial falls in fibrinogen, factor V, and factor VIII and a fall in plasma prothrombin activity of about 15% to 20% of the initial level. Mean plasma factor X activity fell by about 30% of the initial level. Factors participating in the intrinsic coagulation pathway—XII, XI, and IX—all fell by about 50% after injection of a complex made with 16.3 pmol factor Xa and 80 nmol phospholipid per 1 kg body wt and by about 35% after injection of a complex made with 32.6 pmol factor Xa and 40 nmol phospholipid per 1 kg body wt. In contrast, total plasma factor VII activity did not change, and specific plasma factor VIIa levels, which were lower than those measured in human plasma, did not rise after injection of factor Xa/phospholipid. The data are compatible with the hypothesis that factor Xa/phospholipid–induced generation of thrombin in vivo leads to factor XII–dependent activation of the intrinsic pathway of coagulation that results in significant activation of factor X. Further testing of this hypothesis appears warranted.
Platelet Rebound Effect of Alcohol Withdrawal and Wine Drinking in Rats
Authors: Ruf Jean-Claude , Berger Jean-Luc , Renaud Serge
Published: June 2011

AbstractWe investigated in rats fed a purified diet for 2 and 4 months whether wine drinking was associated with the rebound effect on thrombin-induced platelet aggregation observed after alcohol withdrawal. With 6% ethanol drinking or its equivalent in red or white wine, platelet aggregation was reduced similarly by 70% when the animals drank the alcoholic beverages up to the venipuncture. Depriving the rats of alcoholic beverages for 18 hours was associated with an increase in the platelet response of 124% in those receiving 6% ethanol, of 46% with white wine but a decrease of 59% in those with red wine. The protective effect of red wine on platelets could be reproduced by tannins (procyanidins) extracted from grape seeds or red wine and added to 6% ethanol, but not by glycerol or wine without alcohol. That was related to inhibition of the alcohol-induced lipid peroxidation as shown by the lowering of conjugated dienes, lipid peroxides, and the increase in vitamin E in plasma. Owing to tannins, the platelets of rats drinking red wine did not exhibit the rebound effect observed hours after alcohol drinking, eventually associated with sudden death and stroke in humans.
Characterization of LDL and VLDL Binding Sites on Human Basophils and Mast Cells
Authors: Virgolini Irene , Li Shu-Ren , Yang Qiong , Koller Elisabeth , Sperr Wolfgang R. , Angelberger Maria Leimer Peter , Nimpf Johannes , Schneider Wolfgang , Valent Peter
Published: June 2011

AbstractRecent data suggest that basophils and mast cells play a potential role in the processing and accumulation of plasma lipoproteins. This study investigated the interactions of111In-low-density lipoprotein (LDL),111In-acetyl-LDL, and111In-very-low-density lipoprotein (VLDL) with purified primary human blood basophils, immortalized human basophils (KU812 cell line), and a human mast cell line, HMC-1. Binding sites for111In-LDL resolved into curvilinear Scatchard plots indicating two classes of specific binding sites on primary basophils (Bmax1, 7404 sites/cell;Kd1, 1.9 nmol/L; Bmax2, 39 611 sites/cell;Kd2, 29 nmol/L), on KU812 cells (Bmax1, 8290±2690 sites/cell;Kd1, 2.4±0.6 nmol/L; Bmax2, 46 470 sites/cell;Kd2, 33.4±7.8 nmol/L), and on HMC-1 cells (Bmax1, 7840±360 sites/cell;Kd1, 1.8±0.8 nmol/L; Bmax2, 61 450±9900 sites/cell;Kd2, 28.4±9.4 nmol/L). On KU812 cells, binding of111In-LDL was displaced by apolipoprotein (apo)-E–rich high-density lipoprotein (HDL) (IC50, 14±6 nmol/L), LDL (IC50, 29±11 nmol/L), VLDL (IC50, 55±21 nmol/L), HDL2(IC50, 420±140 nmol/L), and heparin (IC50, 67±28 nmol/L), whereas no competition was produced by HDL, HDL3, or acetyl-LDL (IC50, >1 μmol/L). Western blot analysis using the monoclonal antibody C7 confirmed the presence of the LDL receptor on human basophils and HMC-1 cells.111In-acetyl-LDL binding sites (scavenger receptor) could be detected neither on human basophils nor on HMC-1 cells.111In-VLDL bound to a single class of high-affinity binding sites on primary basophils (Bmax, 4320 sites/cell;Kd, 10 nmol/L), KU812 cells (Bmax, 4020±840 sites/cell;Kd, 8±3 nmol/L), and HMC-1 cells (Bmax, 6143±1866 sites/cell;Kd, 4±2 nmol/L).111In-VLDL binding was displaced by VLDL>LDL>apoE-rich HDL but not by heparin (IC50>1 mmol/L). In the presence of prostaglandin E1, the number of111In-LDL receptors increased by 150% (P<.05) in the high-affinity range and by 170% (P<.01) in the low-affinity range, whereas the number of111In-VLDL binding sites remained unchanged. VLDL, LDL, HDL, and the subclasses HDL2and HDL3inhibited immunological histamine release by primary normal basophils (n=3) and mast cells (n=3). Our results provide evidence for the existence of LDL and VLDL binding sites on human basophils and HMC-1 mast cells. The exact biological and pathophysiological roles of these sites remain to be elucidated.
Lack of Role for Nitric Oxide (NO) in the Selective Destabilization of Endothelial NO Synthase mRNA by Tumor Necrosis Factor–α
Authors: Mohamed Farida , Monge Juan Carlos , Gordon Ann , Cernacek Peter , Blais Dominique , Stewart Duncan J.
Published: June 2011

AbstractThe constitutive expression of endothelial nitric oxide (NO) synthase (cNOS) is essential for the physiological regulation of vascular tone and structure. The mechanism of downregulation of steady state cNOS mRNA in human umbilical vein endothelial cells exposed to tumor necrosis factor–α (TNF-α) was investigated by using Northern blot analysis of total cellular RNA. TNF-α produced a dose- and time-dependent decrease in cNOS mRNA expression that was near maximal at 10 U/mL and 6 hours of exposure, respectively. In contrast, steady state expression of endothelin-1 and plasminogen activator inhibitor–1 (PAI-1) mRNA was upregulated by TNF-α. The pharmacological generation of NO using sodium nitroprusside (10 μmol/L) andS-nitroso-acetylpenicillamine (100 to 400 μmol/L) had no effect on cNOS mRNA levels, and TNF-α–induced downregulation of cNOS was not prevented by coincubation with the inhibitors of NO synthesisNω-nitro-l-arginine methyl ester (1 mmol/L) andNG-monomethyll-arginine (10 mmol/L). Under control conditions, cNOS and PAI-1 mRNA were stable after treatment with actinomycin D for periods greater than 24 hours, whereas endothelin-1 message was rapidly degraded (half-life, <1 hour). Pretreatment with TNF-α (30 U/mL) selectively reduced the half-life of cNOS mRNA to less than 12 hours without altering the stability of PAI-1 message. TNF-α–induced destabilization of cNOS mRNA could be partially prevented by coincubation with cycloheximide (1 μmol/L) but was not reproduced by addition of sodium nitroprusside. These findings indicate that TNF-α downregulation of cNOS expression in human endothelial cells results predominantly from the selective destabilization of the mRNA by a mechanism involving the synthesis of new protein. However, NO production by a TNF-α–inducible isoform of NOS did not appear to contribute either to the decrease in steady state cNOS mRNA levels or the shortening of its half-life.
Effects of Exercise Training and Deconditioning on Platelet Function in Men
Authors: Wang Jong-shyan , Jen Chauying J. , Chen Hsiun-ing
Published: June 2011

Abstract
Platelets play an important role in the pathogenesis of cardiovascular disease. It has also been noticed that regular exercise can reduce the risk of cardiovascular disease. This is the first study to demonstrate that endurance exercise training may suppress platelet adhesiveness and aggregation and that deconditioning may reverse the training effects. Healthy male sedentary subjects were randomly divided into control and training groups. The trained men were trained on a bicycle ergometer at about 60% of maximal oxygen consumption for 30 minutes per day, 5 days per week for 8 weeks, then deconditioned for 12 weeks. During the experimental period, blood samples of the trained subjects were collected before and immediately after a progressive exercise test every 4 weeks. The same experiments were applied to the controls at the beginning of this study and 8 weeks thereafter. A tapered parallel-plate chamber was used to assess platelet adhesiveness. Platelet aggregation induced by ADP was evaluated by the percentage of reduction in single platelet count. Our results showed that (1) platelet adhesiveness and aggregability were increased by short-term strenuous exercise in both control and trained groups, but the enhancement of platelet aggregability was decreased after exercise training in the trained subjects; (2) at rest and immediately after strenuous exercise, platelet adhesiveness and aggregability were decreased by training, whereas they were unchanged in the control group; and (3) deconditioning reversed the training effects on resting and postexercise platelet adhesiveness and aggregability back to the pretraining state. These results suggest that platelet adhesiveness and aggregability may be depressed by exercise training but be reversed back to the pretraining state after deconditioning.
Patients With ApoE3 Deficiency (E2/2, E3/2, and E4/2) Who Manifest With Hyperlipidemia Have Increased Frequency of an Asn 291→Ser Mutation in the Human LPL Gene
Authors: Zhang Hanfang , Reymer Paul W. A. , Liu Ming-Sun , Forsythe Ian J. , Groenemeyer Bjorn E. , Frohlich Jiri , Brunzell John D. , Kastelein John J. P. , Hayden Michael R. , Ma Yuanhong
Published: June 2011

AbstractApproximately 1% to 2% of persons in the general population are homozygous for a lipoprotein receptor–binding defective form of apoE (apoE2/2). However, only a small percentage (2% to 5%) of all apoE2/2 homozygotes develop type III hyperlipoproteinemia. Interaction with other genetic and environmental factors are required for the expression of this lipid abnormality. We sought to investigate the possible role of LPL gene mutations in the development of hyperlipoproteinemia in apoE2/2 homozygotes and in apoE2 heterozygotes. As a first step, we performed DNA sequence analysis of all 10 LPL coding exons in 2 patients with the apoE2/2 genotype who had type III hyperlipoproteinemia and identified a single missense mutation (Asn 291→Ser) in exon 6 of the LPL gene. The mutation was then found in 5 of 18 patients with type III hyperlipoproteinemia who had the apoE2/2 genotype (allele frequency=13.9%;P⩽7.4×10−5) and 6 of 22 hyperlipidemic E2 heterozygous patients with the apoE3/2 and E4/2 genotype (allele frequency=13.6%;P=2.2×10−5). In contrast, this mutation was found in only 3 of 230 normolipidemic controls (allele frequency=0.7%). In vitro mutagenesis studies revealed that the Asn 291→Ser mutant LPL had approximately 60% of LPL catalytic activity and approximately 70% of specific activity compared with wild-type LPL. The heparin-binding affinity of the mutant LPL was not impaired. Our data suggest that the Asn 291→Ser substitution is likely to be a significant predisposing factor contributing to the expression of different forms of hyperlipidemia when associated with other genetic factors such as the presence of apoE2.
Mutations in the Gene for Lipoprotein Lipase
Authors: Pimstone Simon N. , Gagné S. Eric , Gagné Claude , Lupien Paul J. , Gaudet Daniel , Williams Roger R. , Kotze Maritha , Reymer Paul W. A. , Defesche Joep C. , Kastelein John J. P. , Moorjani Sital , Hayden Michael R.
Published: June 2011

AbstractFamilial hypercholesterolemia (FH) is characterized by elevated plasma concentrations of LDL cholesterol resulting from mutations in the gene for the LDL receptor. Low HDL cholesterol levels are seen frequently in patients both heterozygous and homozygous for mutations in this gene. Suggested mechanisms for reduced HDL levels in FH patients have been altered apolipoprotein A-1 metabolism and elevated cholesteryl ester transfer protein activity, but the molecular basis for hypoalphalipoproteinemia in any of these patients has not yet been identified. We investigated four large families in which individuals were found to be double heterozygotes for both FH and lipoprotein lipase (LPL) deficiency. These double heterozygotes have significantly less HDL cholesterol than persons with FH or LPL heterozygosity alone. In the double heterozygotes, HDL particle composition is not significantly different from FH heterozygotes, suggesting a quantitative rather than qualitative defect in HDL metabolism in these persons. We propose that mutations in the LPL gene may be a cause of low HDL cholesterol levels in some individuals heterozygous for FH.
Relations Between Plasma Lipids and Postheparin Plasma Lipases and VLDL and LDL Subfraction Patterns in Normolipemic Men and Women
Authors: Tan C. E. , Forster L. , Caslake M. J. , Bedford D. , Watson T. D. G. , McConnell M. , Packard C. J. , Shepherd J.
Published: June 2011

Abstract
VLDL
1
, VLDL
2
, IDL, and LDL and its subfractions (LDL-I, LDL-II, and LDL-III) were quantified in 304 normolipemic subjects together with postheparin plasma lipase activities, waist/hip ratio, fasting insulin, and glucose. Concentrations of VLDL
1
and VLDL
2
rose as plasma triglycerides (TGs) increased across the normal range, but the association of plasma TGs with VLDL
1
showed a steeper slope than that of VLDL
2
(
P
<.001). Plasma TG level was the most important determinant of LDL subfraction distribution. The least dense species, LDL-I, decreased as the level of this plasma lipid rose in the population. LDL-II in both men and women exhibited a positive association with plasma TG level in the range 0.5 to 1.3 mmol/L, increasing from about 100 to 200 mg/dL. In contrast, within this TG range the LDL-III concentration was low (≈30 mg/dL) and changed little. As plasma TGs rose from 1.3 to 3.0 mmol/L there was a significant fall in LDL-II concentration in men (
r
=−.45,
P
<.001) but not in women (
r
=−.1, NS). Conversely, above the TG threshold of 1.3 mmol/L there was a steeper rise in LDL-III concentrations in men than in women (
P
<.001); 42% of the men had an LDL-III in the range associated with high risk of heart disease (>100 mg lipoprotein/dL plasma) compared with only 17% of the women. Other influences on the LDL subfraction profile were the activities of lipases and parameters indicative of the presence of insulin resistance. Men on average had twice the hepatic lipase activity of women. This enzyme was not strongly associated with variation in the LDL subfraction profile in men, but in women it was correlated with LDL-III (
r
=.39,
P
=.001) and remained a significant predictor in multivariate analysis. Increased waist/hip ratio, fasting insulin, and glucose were correlated negatively with LDL-I and positively with LDL-III, primarily, at least in the case of LDL-III, through raising plasma TGs. On the basis of these cross-sectional observations we postulate the following model for the generation of LDL-III. Subjects develop elevated levels of large TG-rich VLDL
1
for a number of reasons, including failure of insulin action. The increase in the concentration of VLDL
1
expands the plasma TG pool, and this, via the action of cholesteryl ester transfer protein (which facilitates neutral lipid exchange between lipoprotein particles), promotes the net transfer of TGs into LDL-II, the major LDL species. A hepatic lipase activity in the male range (due possibly to androgen/estrogen imbalance in women) is then required to lipolyze TG-enriched LDL-II and to generate a concentration of small, dense LDL-III that exceeds the risk limit of 100 mg/dL.
In Vivo Effect of TGF-β1
Authors: Kanzaki Tetsuto , Tamura Ken , Takahashi Kazuo , Saito Yasushi , Akikusa Bunshiro , Oohashi Hideya , Kasayuki Noriaki , Ueda Makiko , Morisaki Nobuhiro
Published: June 2011

Abstract
The in vivo effect of transforming growth factor–β1 (TGF-β1) was studied in a model system in which arterial intimal thickening was induced by injury of rabbit arteries with a balloon catheter (BCI). Intimal area and its ratio to medial area in carotid arteries after BCI were significantly higher in rabbits treated with 10 μg/kg TGF-β1 and 10 mg/kg aspirin IV QD (TGF-β1 group) than in those treated with 10 mg/kg aspirin IV QD only (control group). Intimal cell numbers in the TGF-β1 and control groups were not significantly different from each other, but matrix volume in the intimal layer was significantly higher in the TGF-β1 group. By immunohistochemical and Northern blot analyses, the fibronectin content in carotid intimal and medial layers was greater in the TGF-β1 group compared with that in the control group. Thus, in intimal thickenings induced by BCI, TGF-β1 mainly enhanced the formation of matrix containing fibronectin. Moreover, the mRNAs of TGF-β type I and type II receptors were detected in carotid arteries 7 and 14 days after, but not before, BCI. Thus, TGF-β1 influences the process of intimal thickening induced by BCI through a receptor-mediated mechanism in vivo. The significance of this fact is discussed in relation to the development of atherosclerosis.
HDL3Stimulates Multiple Signaling Pathways in Human Skin Fibroblasts
Authors: Walter Michael , Reinecke Holger , Nofer Jerzy-Roch , Seedorf Udo , Assmann Gerd
Published: June 2011

AbstractThe influence of HDL3on phospholipid breakdown was examined in human skin fibroblasts. HDL3elicited phosphatidylcholine (PC) and phosphatidylinositol (PI) turnover and activated multiple phospholipases. In [14C]lyso-PC–labeled or [14C]choline (Cho)-labeled cells, a biphasic activation of PC-specific phospholipase D (PLD) with peak maxima 30 to 60 seconds and 5 to 7 minutes after stimulation with 20 μg/mL HDL3was shown by (1) a 1.5- to 3-fold increase in [14C]phosphatidic acid (PA), (2) a 1.6-fold increase in Cho release, and (3) transphosphatidylation of PC to phosphatidylbutanol in the presence of 0.3% butanol. Activation of PC-specific PLD was paralleled by an activation of PC-specific phospholipase C (PLC). A significant increase in [14C]diacylglycerol (DG) was seen from 2 minutes after stimulation onward and remained for at least 2 hours. By means of butanol, the PA-phosphohydrolase (PPH) inhibitor propranolol, and the PC-PLC inhibitor D609, we demonstrated that the initial PC-derived DG formation occurred primarily by a coupled PLD/PPH pathway and that a major part of the sustained DG formation was derived directly from PC by PC-PLC. By downregulating protein kinase C (PKC) we demonstrated that PKC activates PC-PLC and desensitizes PC-PLD at longer incubation times. The sustained PC hydrolysis as well as HDL3-mediated PI turnover and PC resynthesis was observed on stimulation with 5 to 75 μg/mL HDL3, whereas the rapid activation of PC-PLD/PPH was detected only on stimulation with HDL3at concentrations of between 10 and 75 μg/mL. Only the latter response could be mimicked by apolipoprotein A-I and apolipoprotein A-II proteoliposomes, and only this response was inducible by cholesterol loading. The HDL3-mediated second-messenger responses were inhibited by modification of HDL3by tetranitromethane and could not be mimicked by protein-free liposomes. These data suggest that HDL3-induced cell signaling in human skin fibroblasts is mediated by specific protein-receptor interaction and that more than one agonist activity may be involved.
The Interstitium of the Human Arterial Wall Contains Very Large Amounts of Extracellular Superoxide Dismutase
Authors: Strålin Pontus , Karlsson Kurt , Johansson Bengt O. , Marklund Stefan L.
Published: June 2011

Abstract
The levels of the secreted, interstitially located extracellular superoxide dismutase (EC-SOD), the cytosolic copper-and-zinc–containing SOD (CuZn-SOD), and the mitochondrial manganese-containing SOD (Mn-SOD) were measured in the walls of human coronary arteries, proximal thoracic aortas, and saphenous veins. The blood vessel walls, particularly the arteries, were found to contain exceptionally large amounts of EC-SOD, whereas the levels of CuZn-SOD and Mn-SOD were relatively low compared with other tissues. Analysis of EC-SOD by immunohistochemistry indicates an even distribution in the vessel wall, including large amounts in the arterial intima. Arterial smooth muscle cells were found to secrete large amounts of EC-SOD and likely are the principal source of the enzyme in the vascular wall. The EC-SOD concentration in the human arterial wall extracellular space is high enough to efficiently suppress the putative pathological effects of the superoxide radical, such as oxidation of LDL and reaction with nitric oxide to form the deleterious peroxynitrite. The levels of EC-SOD in the aortic wall are found to vary widely among species and were on average 6440 U/g in humans, 4340 U/g in the cow, 2660 U/g in the pig, 160 U/g in the dog, 770 U/g in the cat, 2390 U/g in the rabbit, 90 U/g in the rat, and 3400 U/g in the mouse. There were only moderate differences in the amounts of CuZn-SOD and Mn-SOD. This wide variation in EC-SOD content suggests that the susceptibility to pathologies induced by superoxide radicals in the vascular wall interstitium should vary widely among species.
A Prospective, Randomized Trial of Phenytoin in Nonepileptic Subjects With Reduced HDL Cholesterol
Authors: Miller Michael , Burgan Rachel G. , Osterlund Laura , Segrest Jere P. , Garber David W.
Published: June 2011

AbstractObservational studies have demonstrated a positive association between phenytoin use and HDL cholesterol (HDL-C). Our goal was to determine whether phenytoin raises HDL-C in nonepileptic subjects at risk for coronary artery disease. We performed a double-blind, placebo-controlled, parallel-group study in 41 subjects with reduced levels of HDL-C. Subjects were placed on an American Heart Association Step I diet and were randomized to receive either phenytoin or placebo for 3 months. Serum levels of phenytoin were monitored and adjusted to between 7.5 and 15 μg/mL. Fasting levels of lipids and lipoproteins were determined twice at baseline (weeks −2 and −1) and during the treatment phase of the study (weeks 11 and 12). Compared with dietary baseline, phenytoin-treated subjects experienced significant paired percent increases in total HDL-C (12.4%;P<.01), an effect confined to the HDL2subfraction (137%;P<.01). The paired percent increases in HDL-C and HDL2levels remained significant after adjustment for placebo (P<.05,P<.025, respectively). There were no significant differences in the paired percent changes from dietary baseline in total cholesterol, triglyceride, or LDL cholesterol levels between placebo and phenytoin-treated groups. The significant paired percent increases in total HDL-C and HDL2from dietary baseline suggest a potential role for phenytoin in subjects with reduced levels of HDL-C.
Familial Hypobetalipoproteinemia Is Not Associated With Low Levels of Lipoprotein(a)
Authors: Averna Maurizio , Marcovina Santica M. , Noto Davide , Cole Thomas G. , Krul Elaine S. , Schonfeld Gustav
Published: June 2011

Abstract
To assess whether very low concentrations of LDL affected lipoprotein(a) [Lp(a)] concentrations and apo(a) associations with lipoproteins, we studied Lp(a) levels and associations in heterozygous subjects with familial hypobeta-lipoproteinemia FHBL) associated with several truncated forms of apoB-100, ranging from apoB-31 to apoB-89. Distributions of apo(a) isotypes were assessed by a combined electrophoresis-immunoblotting procedure that detects 34 isoforms. Lp(a) concentrations were quantified by two ELISAs, one detecting total apo(a) and the other apoB-bound apo(a) in plasma. Associations of apo(a) with plasma lipoproteins were evaluated by gel permeation chromatography (FPLC) and DGUC followed by analyses of elution and gradient fractions by apo(a) ELISA. In addition, associations were examined by nondenaturing electrophoresis or immunoprecipitation of whole plasma and examination of contents by immunoblotting. Finally, interactions between r-apo(a) and LDLs were evaluated in reconstitution experiments. The distributions of apo(a) isotypes did not differ between FHBL-affected and unaffected members of the same kindreds, and concentrations of Lp(a) were similar even when subjects were matched for isotypes both within and across kindreds. In subjects heterozygous for apo(a) isoforms, the smaller isoforms were inversely related to Lp(a) concentrations, the larger isoforms were not. The regression lines between Lp(a) concentrations and the smaller apo(a) isoforms were significant and negative in slope for both FHBL-affected and unaffected subjects, but the slopes of the lines did not differ. In multiple regression analyses, only the sizes of the smaller apo(a) isoforms contributed to the prediction of Lp(a) concentrations. ApoB-size made no difference. In simple apoB-100/apoB-truncation heterozygotes, virtually all apo(a) was complexed with apoB-100–containing particles but not apoB-truncation particles, and r-apo(a) recombined with apoB-100–containing LDLs but not with apoB-89–containing LDLs. Thus, (1) low apoB levels do not affect the plasma concentrations of Lp(a), (2) apo(a) binds apoB-100 to form Lp(a) particles of usual sizes and densities, and (3) apoB truncations even as large as apoB-89 do not form covalent bands with apo(a), although noncovalent associations with apoB-89 may be present in plasma.
A Monoclonal Antibody That Recognizes the GPIIb/IIIa Antagonist DMP 728
Authors: Reilly Thomas M. , Mousa Shaker A. , Racanelli Adrienne L. , Thoolen Martin J. , Flint Sandra K. , Bozarth Jeffrey M. , Mu Dun X. , Walton Harry L.
Published: June 2011

Abstract
Since hemorrhagic events represent a major safety concern associated with the use of new antithrombotic therapies such as glycoprotein (GP) IIb/IIIa receptor blockade, we evaluated the ability of a monoclonal antibody recognizing DMP 728 (cyclic [
d
-2-aminobutyryl-N2-methyl-
l
-argininyl-glycyl-
l
-aspartyl-3-aminomethyl-benzoic acid] methanesulfonic acid salt), a potent GPIIb/IIIa receptor antagonist, to reverse the pharmacological actions of DMP 728 in the dog. DC11 was chosen for in vivo evaluation based on its ability to inhibit the binding of [
3
H]DMP 728 to activated platelets and to attenuate the inhibition of ADP-induced aggregation on platelet-rich plasma ex vivo by DMP 728. After anesthesia mongrel dogs were given DMP 728 (20 μg/kg body wt IV) infused into the femoral vein, bleeding times were determined using a Simplate device from incisions on the backside of the tongue, and platelet aggregation was determined ex vivo. Nearly complete inhibition of platelet aggregation was observed for the dogs treated with DMP 728 (20 ug/kg IV) for up to 210 minutes, and bleeding times were prolonged >15 minutes for 2 hours and remained elevated for more than 4 hours. DC11 (0.2 or 1.0 mg/kg body wt IV) given to dogs 10 minutes after DMP 728 resulted in 50% attenuation of the effect of DMP 728 on aggregation at 3 hours. Approximately 34% inhibition of the DMP 728–mediated bleeding time was achieved at 1 hour with the 0.2 mg/kg dose, whereas approximately 50% inhibition of the bleeding time was observed for the 1 mg/kg dose at 1 hour. The bleeding time for the higher dose of DC11 returned to control levels at 2 hours, whereas the lower dose of DC11 returned to control levels at 3 hours. These results suggest that monoclonal antibody DC11 may have potential utility as an antidote to DMP 728 in neutralizing any unexpected bleeding complications associated with its use.
Distribution and Correlates of Insulin in Elderly Men
Authors: Burchfiel Cecil M. , Curb J. David , Sharp Dan S. , Rodriguez Beatriz L. , Arakaki Richard , Chyou Po-Huang , Yano Katsuhiko
Published: June 2011

Abstract
The role of insulin in cardiovascular disease is uncertain, and studies in elderly or minority populations are infrequent. Fasting and 2-hour insulin concentrations and their cross-sectional associations with cardiovascular risk factors were examined in 3562 elderly (aged 71 to 93 years) Japanese American men from the Honolulu Heart Program who were reexamined between 1991 and 1993. Insulin distributions were skewed (mean and median: 16.8 and 12 μU/mL for fasting; 117.2 and 93 μU/mL for 2-hour); fasting but not 2-hour insulin levels declined significantly with age (
P
<.0001 and
P
=.54, respectively). Factors most strongly correlated with insulin included measures of obesity, fat distribution, and levels of triglyceride, glucose (
r
=.38 to
r
=.50 fasting,
r
=.21 to
r=
.27 2-hour), and HDL cholesterol (
r
=−.41 and
r
=−.22, respectively). Other correlates included fibrinogen, hematocrit, heart rate, blood pressure, cigarettes per day (all positive), alcohol, physical activity, and forced vital capacity (negative). Associations were also evident across risk factor quintiles. Insulin levels were significantly elevated in men with hypertension and diabetes. In multiple linear regression analyses, log
10
fasting insulin was positively and independently associated with body mass index, triglycerides, glucose, fibrinogen, hematocrit, heart rate, diabetes, and hypertension and negatively associated with HDL cholesterol, physical activity, and forced vital capacity. In general, results were similar for log
10
2-hour insulin and when subjects who fasted <12 hours or had diabetes were excluded. Substitution of medication use and blood pressure for hypertension indicated independent associations of medication use but not blood pressure with insulin. These findings suggest that fasting and 2-hour insulin levels are associated with several key features of insulin resistance syndrome in elderly Japanese American men.
Human Vascular Smooth Muscle Cell–Monocyte Interactions and Metalloproteinase Secretion in Culture
Authors: Lee Elaine , Grodzinsky Alan J. , Libby Peter , Clinton Steven K. , Lark Michael W. , Lee Richard T.
Published: June 2011

AbstractDegradation of the atherosclerotic plaque extracellular matrix could destabilize the lesion, rendering it more prone to rupture. Both macrophages and vascular smooth muscle cells (SMCs) are potential sources of matrix metalloproteinases (MMPs), secreted enzymes that can digest vascular matrix. We explored interactions between human vascular SMCs and human monocytes that result in the secretion of interstitial collagenase (MMP-1) and stromelysin (MMP-3). Monocytes alone or those treated with SMC-conditioned media did not secrete these metalloproteinases as detectable by Western blot analysis. SMCs increased secretion of both MMP-1 and MMP-3 greater than 20-fold when cocultured with monocytes or when treated with monocyte-conditioned media. Addition of macrophage colony stimulating factor (≤1000 U/mL) to cocultures of monocytes and SMCs did not affect metalloproteinase secretion. Recombinant interleukin (IL)-1 receptor antagonist inhibited MMP-1 and MMP-3 induction in SMC cultures treated with monocyte-conditioned media (94% and 96% reduction, respectively), while a neutralizing antibody to tumor necrosis factor-α had no significant effect on metalloproteinase secretion. In contrast to the induction by monocyte-conditioned media of MMP-1 and MMP-3 secretion by SMCs, monocyte-conditioned media did not increase secretion of 72-kD gelatinase (MMP-2). Thus, monocytes induce MMP-1 and MMP-3 secretion by vascular SMCs through an IL-1–dependent mechanism. This response of SMCs to a defined macrophage product may contribute to plaque destabilization by mononuclear phagocytes in the lesion.
Characterization of a Splice-Site Mutation in the Gene for the LDL Receptor Associated With an Unpredictably Severe Clinical Phenotype in English Patients With Heterozygous FH
Authors: Sun X.-M. , Patel D.D. , Bhatnagar D. , Knight B.L. , Soutar A.K.
Published: June 2011

Abstract
We have identified a substitution of G to A in the first base pair of intron 3 in the LDL receptor gene of an English heterozygous familial hypercholesterolemia (FH) patient. Reverse transcription, amplification, and nucleotide sequencing of the LDL receptor mRNA from mononuclear blood cells showed both the normal mRNA and one that lacked the nucleotides encoded by exon 3, which codes for repeat 2 of the ligand-binding domain. The same mutant allele was identified in 2/200 unrelated FH patients from the London area and 4/77 from Manchester. Immunoblotting of cultured lymphoblasts from the index patient revealed the normal receptor protein and smaller amounts of a receptor protein with electrophoretic mobility consistent with a deletion of the 41 amino acid residues encoded by exon 3. Normal amounts of a similar protein were observed when the mutant cDNA was expressed in heterologous cells; this protein showed reduced binding affinity for LDL but bound apoprotein E–containing lipoproteins normally. Despite these and other observations that repeat 2 of the binding domain is relatively unimportant for receptor function in vitro, carriers of this allele exhibit a severe clinical phenotype, typical of FH. Thus, the relationship between genotype and phenotype in heterozygous FH is not always predictable.
Redox Status and Protein Binding of Plasma Homocysteine and Other Aminothiols in Patients With Early-Onset Peripheral Vascular Disease
Authors: Mansoor M. Azam , Bergmark Claes , Svardal Asbjørn M. , Lønning Per Eystein , Ueland Per M.
Published: June 2011

Abstract
Elevated total homocysteine (Hcy) in plasma is an independent risk factor for early-onset vascular disease in the coronary, cerebral, and peripheral arteries. Different forms of Hcy, and their relation to other aminothiols in plasma, have not been investigated in patients with vascular disease. We therefore investigated 65 patients (35 men and 30 women) operated on for peripheral arterial disease at <50 years of age and 65 age- and sex-matched control subjects. Total, reduced, oxidized, and protein-bound Hcy, cysteine (Cys), and cysteinylglycine (CysGly) were measured 0 to 11 years (mean, 6 years) postoperatively, in the fasting state, and after a standard methionine loading dose that caused a transient increase in reduced, oxidized, and protein-bound Hcy. All forms of Hcy and Cys, except reduced Cys, were higher in fasting patients than fasting control subjects. A similar difference between the groups was observed after methionine loading. The levels of most Hcy forms both during fasting and after methionine loading were related to smoking, but multivariate analysis showed that the difference between patients and control subjects could not be explained by smoking alone. Notably, reduced Cys and the reduced/total ratio for Cys were significantly higher in control subjects than in patients, both during fasting and after methionine loading. In both groups, the redox status and protein binding of the various aminothiols in plasma were interactive, as demonstrated by positive correlations between their reduced/total ratios and by a decrease in protein-bound Cys when protein-bound Hcy was elevated during methionine loading. Serum folate and to a lesser degree serum cobalamin and vitamin B
6
were predictors of oxidized and protein-bound Hcy in some patients and control subject subgroups. Thus, reduced, oxidized, and protein-bound Hcy are elevated and reduced Cys is decreased in patients with peripheral arterial disease. Reduced Hcy acts as a pro-oxidant in vitro and is a possible atherogenic agent, whereas reduced Cys may be a protective agent as a part of the antioxidant defense system. The protein binding and redox status of different plasma aminothiols are interactive in a way suggesting ongoing redox cycling and disulfide exchange reactions. Thus, Hcy is one component in a complex system.
Inhibition of Thromboxane Biosynthesis and Platelet Function by Simvastatin in Type IIa Hypercholesterolemia
Authors: Notarbartolo Alberto , Davı̀ Giovanni , Averna Maurizio , Barbagallo Carlo M. , Ganci Antonina , Giammarresi Carlo , Placa Francesco P. La , Patrono Carlo
Published: June 2011

Abstract
Thromboxane A
2
(TXA
2
) biosynthesis is enhanced in the majority of patients with type IIa hypercholesterolemia. Because simvastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) was previously shown to reduce platelet aggregation and TXB
2
production ex vivo, we investigated TXA
2
biosynthesis and platelet function in 24 patients with type IIa hypercholesterolemia randomized to receive in a double-blind fashion simvastatin (20 mg/d) or placebo for 3 months. The urinary excretion of 11-dehydro-TXB
2
, largely a reflection of platelet TXA
2
production in vivo, was measured by a previously validated radioimmunoassay technique. Blood lipid levels and urinary 11-dehydro-TXB
2
excretion were significantly (
P
<.001) reduced by simvastatin. In contrast, placebo-treated patients did not show any statistically significant changes in either blood lipids or 11-dehydro-TXB
2
excretion. The reduction in 11-dehydro-TXB
2
associated with simvastatin was correlated with the reduction in total cholesterol (
r
=.81,
P
<.0001), LDL cholesterol (
r
=.79,
P
<.0001), and apolipoprotein B (
r
=.76,
P
<.0001) levels. Platelets from patients with type IIa hypercholesterolemia required significantly (
P
<.01) more collagen and ADP to aggregate and synthesized less TXB
2
in response to both agonists after simvastatin therapy. Bleeding time, platelet sensitivity to Iloprost, and blood lipoprotein(a) and HDL cholesterol levels were not significantly affected by either treatment. We conclude that enhanced TXA
2
biosynthesis in type IIa hypercholesterolemia is, at least in part, dependent on abnormal cholesterol levels and/or other simvastatin-sensitive mechanisms affecting platelet function.
Effects of 26-Aminocholesterol, 27-Hydroxycholesterol, and 25-Hydroxycholesterol on Proliferation and Cholesterol Homeostasis in Arterial Myocytes
Authors: Corsini A. , Verri D. , Raiteri M. , Quarato P. , Paoletti R. , Fumagalli R.
Published: June 2011

AbstractThe major relation existing between cell growth and cholesterol homeostasis prompted us to investigate the effect of 26-aminocholesterol (26-NH2), 27-hydroxycholesterol (27-OH), and 25-hydroxycholesterol (25-OH) on these cellular events. To test this relation, we incubated human and rat arterial myocytes with the sterols for 72 hours. All the tested compounds (0.5 to 7.5 μmol/L) inhibited rat and human myocyte proliferation and cholesterol biosynthesis in a dose-dependent manner. 26-NH2was more potent than oxysterols in inhibiting human myocyte proliferation but equieffective in rat cells; 27-OH and 25-OH displayed similar activity in both cell lines. Inhibition of nuclear incorporation of thymidine in rat myocytes is consistent with decreased cell count. The antiproliferative effect of the tested sterols was reversible. The high inhibition (80%) of cholesterol biosynthesis necessary to induce a decrease in myocyte proliferation suggests a causal relation between the cholesterol synthetic pathway and these cellular processes. In addition, all the tested sterols were able to inhibit hydroxymethyl glutaryl–coenzyme A reductase activity in intact myocytes but not in cell-free extracts. The finding that 26-NH2but not 27-OH or 25-OH does not suppress LDL receptor activity in either human or rat myocytes supports the achievement of selectivity over the coordinately regulated LDL receptor gene. The ability of 26-NH2to interfere with myocyte proliferation and cholesterol synthesis without affecting the LDL receptor pathway confers at least in vitro a pharmacological interest on the compound in the process of atherogenesis.